Protein labeling with cyanobenzothiazole conjugates

ABSTRACT

The invention provides compounds and methods for site-specifically labeling proteins with cyanobenzothiazole derivatives of formula I. For example, the invention provides methods for labeling the N-terminus of a protein that terminates with a cysteine residue. The invention also provides methods for isolating an N-terminally labeled protein and methods for detecting an N-terminally labeled protein.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/383,832 filed on Mar. 26, 2009, now U.S. Pat. No. 9,459,249, whichclaims priority to U.S. Provisional Application No. 61/040,073 filed onMar. 27, 2008, which are incorporated herein by reference.

BACKGROUND

Site-specific labeling of biomolecules with fluorophores often requirescareful choice of labeling chemistry, optimization of the labelingreaction and characterization of the labeled biomolecules for labelingefficiency, site-specificity, and retention of functionality. The twomost commonly used approaches for proteins are based on chemicalcoupling to sulfhydryl groups or primary amines, which result indifferent labeling patterns on different proteins as a consequence ofthe unique content and distributions of cysteine (Cys) and lysineresidues in a given protein. The most common method for site-specificlabeling of proteins with fluorophores is Cys-specific labeling withthiol-reactive reagents. During this reaction, proteins withsurface-exposed Cys residues are covalently modified by maleimide,iodoacetamide, or other reactive conjugates of fluorophores (Waggoner,Methods Enzymol., 246:362 (1995); Haugland, Handbook of FluorescentProbes and Research Products, 8th ed., 2002; Selvin, Methods Enzymol.,246:300 (1995)). This is a method of choice for small proteins (< about200 residues) because cysteine is a rare amino acid and can besubstituted easily with other amino acids using site-directedmutagenesis (Kunkel et al., Methods Enzymol., 205:125 (1991)).

If a protein of interest has no Cys residues, the site of incorporationof the label is selected after inspection of a high-resolutionthree-dimensional structure (generated using x-ray crystallography ornuclear magnetic resonance). Labeling should not perturb the enzymaticactivity or the spatial arrangement of the protein sequence (also knownas the “protein fold”). Subsequently, an existing amino acid (preferablyhaving a side chain of charge, size, and hydrophobicity similar to thatof Cys) at the site of choice is substituted by a Cys usingsite-directed mutagenesis (Kunkel et al., 1991).

If an unmodified protein has a single preexisting Cys, structuralinformation, along with measurements of the surface accessibility of Cysside chain (Kapanidis et al., J. Mol. Biol., 312:453 (2001)), maydetermine whether the existing Cys can be used for labeling; otherwise,the preexisting Cys can be converted to the structurally similar aminoacid serine, and the procedure for Cys-free proteins can be followed.

In a recently developed approach referred to as expressed proteinligation (EPL) (Muir, Annu. Rev. Biochem., 72:249 (2003)), proteins areexpressed in C-terminal fusion with an intein domain and an affinitytag. The resulting fusion proteins can be separated from the proteins ofthe expression host on an affinity matrix. Treatment of the immobilizedprotein with a high concentration of thiol leads to the cleavage of thepeptide bond between intein and target protein. The cleaved proteincarries a thioester group on the C-terminus that can be coupled to apeptide (or, in fact, any molecule) bearing a Cys at its N-terminus bynative chemical ligation to generate a native peptide bond at thecoupling site (Dawson et al., Science, 266:776 (1994)). Although thisapproach was successfully used for protein engineering, its shortcomingsare related to the necessity of expressing a large fusion protein thatmay influence the solubility and folding of the target protein and thedifferent efficiencies of intein splicing due to the influence of theflanking residues of the target protein (Zhang et al., Gene, 275:241(2001)).

An alternative strategy is where a thioester-conjugated functionalitysuch as a fluorophore is coupled onto the N-terminal Cys of arecombinant protein (Schuler et al., Bioconjugate Chem., 13:1039(2002)). In some cases, Cys on position 2 becomes N-terminal uponmethionine cleavage by aminopeptidase of the expression host, althoughthe efficiency varies among proteins (Gentle et al., Bioconjugate Chem.,15:658 (2004)). In another strategy, an N-terminal Cys is created byself-cleavage of an intein domain fused N-terminally to the targetprotein. Alternatively, the N-terminal Cys residue can be generated byproteolytic cleavage of a properly engineered protease cleavage site.Among proteases that were shown to tolerate Cys at the +1 position ofthe cleavage site are factor X, Precision protease, and TEV proteases(Cotton et al., Chem. Biol., 7:253 (2000); Tolbert et al., Angew. Chem.Int. Ed., 41:2171 (2002)).

Accordingly, there is a need for compounds, compositions, and methods toaid in site-specifically linking chemical groups onto specific sites ofproteins or peptides.

SUMMARY OF THE INVENTION

The invention provides cyanobenzothiazoles linked to fluorophores orother detectable groups, e.g., reporter moieties, affinity moieties,antigens, quencher compounds, photocrosslinking moieties, or solidsupports, that may aid in the identification, quantification and/orpurification of biomolecules, such as proteins. For instance, suchcyanobenzothiazole derivatives are capable of rapid and specificreaction with proteins containing a cysteine (“Cys”) residue at theN-terminus. By reacting with proteins having an N-terminal Cys residue,the cyanobenzothiazole derivatives can introduce a reporter moiety orother functionality at the N-terminus of a protein. In one embodiment,the reaction can proceed by nucleophilic attack of the Cys thiol on thecyano group of the cyanobenzothiazole derivative, followed byconcomitant cyclization.

Cyclization provides a stable thiazoline ring, thereby covalentlyattaching the benzothiazole derivative to the N-terminus of a protein ofinterest. The derivative can include at least one reporter or affinitymoiety covalently linked, for example to the 6′ position, of acyanobenzothiazole. The addition of cyanobenzothiazole derivatives tointernal Cys side chains may be readily eliminated. Elimination may beachieved by addition of a Cys solution and incubation, typically forabout 1 to about 10 minutes, often about 2 to about 5 minutes.

The invention also provides methods of introducing a reporter moiety orother functionality at the N-terminus of a protein of interest using acyanobenzothiazole derivative of the invention, thereby providing alabeled protein. The Cys containing protein may be a protein having orengineered to have a Cys at position 2, may be prepared byintein-mediated methods, or prepared with an appropriate protease. Inone embodiment, the derivative has the following general structureX-L-M, where X is a reporter moiety or an affinity moiety, L is anoptional linker, and M is a cyanobenzothiazole. In one embodiment, L isphotocleavable. In one embodiment, L is a recognition site for anenzyme. In one embodiment, L is not photocleavable. In one embodiment, Lis not a recognition site for an enzyme. In one embodiment, X-L-M is notdetectable but the product of a reaction between X-L-M and a N-terminalCys containing protein is detectable. In one embodiment, X-L-M isdetectable but the product of a reaction between X-L-M and a N-terminalCys containing protein is not detectable. In another embodiment, X-L-Mand the product of a reaction between X-L-M and a N-terminal Cyscontaining protein may be distinguished, e.g., optically.

Application of the methods include but are not limited to site-specificlabeling of proteins for detection or for analysis of protein structureand function. The invention also provides methods for performing anassay to detect molecules such as enzymes of interest using derivativesof 2-cyanobenzothiazole. Enzymes of interest include but are not limitedto kinases, phosphatases, peroxidases, sulfatases, peptidases,glycosidases and proteases, for example, proteases involved inapoptosis. The invention further provides novel compounds andcompositions that can be used in such an assay.

The invention thus provides, according to certain embodiments, an invitro method to N-terminally label proteins. The method includescontacting a mixture, for instance, a protein extract (lysate), purifiedprotein, or components of a protein synthesis system, which mixtureincludes at least one protein with a terminal Cys, for example, anN-terminal Cys, with a derivative of cyanobenzothiazole, which includesat least one reporter moiety or affinity moiety. The cyanobenzothiazolederivative can include at least one reporter or affinity moiety attachedto the benzo moiety of the cyanobenzothiazole. For example, a reporteror affinity moiety can be attached to the 4′, 5′, 6′, or 7′ position ofa cyanobenzothiazole. In certain embodiments, the reporter or affinitymoiety is attached the 6′ position of a cyanobenzothiazole.

In one embodiment, the mixture that contains the terminal Cys protein isa cellular lysate, for instance, a cellular lysate from a commerciallibrary. In one embodiment, the mixture may include at least one nucleicacid molecule, e.g., an mRNA, as well as tRNAs, amino acids and/orcharged tRNAs, ribosomes, one or more initiation factors, one or moreelongation factors, and one or more termination factors, e.g., a proteinsynthesis system. The mixture may also be a combined eukaryotictranscription/translation mixture.

The cyanobenzothiazole derivatives may be employed with any in vitrotranslation system, e.g., eukaryotic translation systems including, butnot limited, to wheat germ extract, an insect cell lysate, a rabbitreticulocyte lysate, prokaryotic (e.g., S30) E. coli, a frog oocytelysate, a dog pancreatic lysate, a human cell lysate, or mixtures ofpurified or semi-purified eukaryotic translation factors. In oneembodiment, proteins having a reporter or affinity moiety at theirN-terminus are then detected, isolated and/or quantitated.

One embodiment provides a method to specifically attach a chemical groupof choice (e.g., a fluorophore or other detectable group) to theC-terminal end of a protein, peptide, or other carboxyl-containingmolecule. The methods include contacting a mixture having the moleculeto be labeled with a benzothiazole derivative, for example, a compoundof formula I. Less stable derivatives can optionally be removed with achase reagent, thereby yielding a mixture having a molecule with astable detectable group.

Benzothiazole compounds of the invention include compounds of formula I:

The variable Z can be H, F, Cl, Br, I, CN, amino, alkylamino,dialkylamino, alkyl ester (e.g., —CO₂(alkyl)), carboxy, carboxylic acidsalt, alkyl amide (—C(═O)NH(alkyl)), phosphate (—OPO(OH)₂), alkylphosphonate, sulfate (—OSO₃H), alkyl sulfonate, nitro, or (C₁-C₁₀)alkyloptionally unsaturated and optionally substituted with amino, hydroxy,oxo (═O), nitro, thiol, or halo. The group Z can be located at the 4′,5′, or 7′ position of the cyanobenzothiazole. In certain embodiments, Zis located at the 7′ position.

Each R¹ can independently be H, F, Cl, Br, I, CN, (C₁-C₆)alkyl,(C₁-C₆)alkoxy, or (C₁-C₆)alkylthio, wherein each alkyl, alkoxy, oralkylthio is optionally substituted with F, Cl, Br, I, amino, alkenyl,alkynyl, cycloalkyl, aryl, alkyl sulfonate, or CO₂M wherein M is H, anorganic cation, or an inorganic cation; wherein n is 0, 1, or 2. Thegroup or groups R¹ can be located at the 4′, 5′, or 7′ position of thecyanobenzothiazole. In certain embodiments, Z can be located at the 7′position.

The group Y can be a linking group comprising (C₁-C₁₆)alkyl optionallysubstituted with one or more (e.g., 1, 2, 3, 4, 1-5, or 1-6) halo,hydroxy, oxo, (C₁-C₆)alkyl, or (C₁-C₆)alkoxy, and optionally interruptedwith one or more (e.g., 1, 2, 3, 4, 1-5, or 1-6) N(R¹), O, S, or—N—C(═O)— groups, or Y can be absent. The term “optionally interrupted”means that one or more, e.g., 1, 2, 3, 4, 1-5, or 1-6, carbon atoms ofthe linking group, including one or both terminal carbons of the linkinggroup, can be replaced with an O, N(R¹), S, or —N—C(═O— group. In someembodiments, Y can optionally be absent, for example, when X is azido(N₃). For example, in some embodiments, Y can be —(C₁-C₆)alkyl-,—O—(C₁-C₆)alkyl-, —O—(C₁-C₆)alkyl-O—, —O—(C₁-C₆)alkyl-NH—,—O—(C₁-C₆)alkyl-(CO)NH—, —NH—(C₁-C₆)alkyl-NH—, —NH—(CO)(C₁-C₆)alkyl-NH—,—NH—(CO)(C₁-C₆)alkyl-(CO)—NH—, or —O—(C₁-C₆)alkyl-(CO)NH—(C₁-C₆)alkyl-.

The group X can be a reporter moiety, an affinity moiety, a quencher, aphotocrosslinking moiety, a solid support, N₃, H, or OH. In certainembodiments, when X is H or OH, the compound of formula I comprises aradioactive moiety or an isotopic variant of any atom other than thecarbon or nitrogen atom of the 2-nitrile moiety, for example, when Y isabsent.

The functionality attached to the cyanobenzothiazole may be any molecule(or portion thereof) that is detectable or capable of detection, orcapable of isolation. Those moieties include, but are not limited to, anucleic acid molecule, i.e., DNA or RNA, e.g., an oligonucleotide, adrug, a protein, a peptide, for instance, an epitope recognized by aligand, a hapten, e.g., keyhole limpet hemacyanin (KLH), a carbohydrate,biotin, a resin, a substrate for an enzyme, a fluorophore, achromophore, and the like, or a combination thereof. For example, anucleic acid reporter moiety can be detected by hybridization,amplification, binding to a nucleic acid binding protein specific forthe nucleic acid reporter, enzymatic assays (e.g., if the nucleic acidmolecule is a ribozyme), or, if the nucleic acid molecule itselfcomprises a molecule which is detectable or capable of detection, forinstance, a radiolabel or biotin, it can be detected by an assaysuitable for that molecule.

A nucleic acid reporter may be useful to detect and/or isolate proteinsin microarrays or ribosomal display. Immuno-PCR and immuno-detection byamplification with T7 polymerase (IDAT) are also amenable to detect anucleic acid reporter. For instance, a nucleic acid reporter attached toa cyanobenzothiazole is employed to label N-terminal Cys containingproteins, the nucleic acid is amplified, e.g., in the presence offluorescent nucleotides, and then the amplified nucleic acid is detected(see published U.S. application 2002/0028450 (Greene et al.)). Protein-or peptide-based reporter or affinity moieties can be detected by ligandbinding, e.g., binding to an antibody specific for the protein orpeptide, or biochemical, enzymatic or luminescent activity, e.g., theprotein based moiety may be a transport domain, an antibody, a caspase,a luciferase or green fluorescent protein (GFP).

Affinity moieties and their corresponding ligands, for instance, maltoseand maltose binding protein, biotin and avidin or streptavidin and a Histag and a metal, such as cobalt, zinc, nickel or copper, find particularuse in protein detection and isolation, e.g., on a solid support such asa bead, resin, or well of a multi-well plate. A fluorescent (orbioluminescent) reporter, such as one detectable by UV and/or visibleexcited fluorescence detection placed on the N-terminus of a protein maybe used to sense changes in a system, like phosphorylation, in realtime. Moreover, a fluorescent molecule, such as a chemosensor of metalions, e.g., a 9-carbonyl-anthracene modified glycyl-histidyl-lysine(GHK) for Cu²⁺, or a pair of fluorescent molecules, e.g., fluoresceinand rhodamine, may be employed to label proteins so as to form proteinbiosensors.

A bioluminescent or fluorescent reporter, such as BODIPY, rhodaminegreen, GFP, or infrared dyes, also finds use in interaction studies,e.g., using BRET, FRET, LRET or electrophoresis, e.g., capillaryelectrophoresis. For interaction studies, one or more specific moleculesare combined with the labeled proteins (either before or afterisolation) to form a mixture containing labeled proteins and one or moremolecules.

The interaction of one or more labeled proteins with one or moremolecules can then be detected. Thus, a derivative of cyanobenzothiazolemay be used in protein synthesis or mixtures of synthesized protein todetect, isolate and quantitate such proteins, e.g., human proteins,viral proteins, bacterial proteins, and parasitic proteins, includingrecombinant gene products, gene fusion products, enzymes, cytokines,hormones, immunogenic proteins, carbohydrate binding proteins, lipidbinding proteins, nucleic acid binding proteins, and fragments thereof.

Any protein, encoded by a naturally occurring or recombinant gene, maybe labeled with a moiety using the methods of the invention. Proteinscontaining the moiety can then be detected and/or isolated by methodsknown in the art. For example, proteins can be detected and/or isolatedby taking advantage of unique properties of the moiety, e.g., thespecific spectral property, of the moiety, by any means includingelectrophoresis, gel filtration, high-pressure or fast-pressure liquidchromatography, mass spectroscopy, affinity chromatography, ion exchangechromatography, chemical extraction, magnetic bead separation,precipitation, hydrophobic interaction chromatography (HIC), or anycombination thereof. The isolated proteins may be employed forstructural and functional studies, for the development of diagnosticapplications, for the preparation biological or pharmaceutical reagents,as a tool for the development of drugs, and for studying proteininteractions or for the isolation and characterization of proteincomplexes.

The invention also contemplates kits. In one embodiment, the kitcomprises a derivative of cyanobenzothiazole having a reporter oraffinity moiety and optionally one or more reagents for the detection,identification, and/or purification of labeled proteins, for example,reagents such as beads, a resin, a column, and the like. In anotherembodiment, the kit comprises a derivative of cyanobenzothiazole and atleast one reagent. In certain embodiments, the reagent and thederivative are in separate containing means (e.g., tubes, vials, and thelike). Some kits include an immobilized derivative ofcyanobenzothiazole, or reagents to immobilize the cyanobenzothiazolederivative. Such kits are useful to prepare one or more N-terminallylabeled proteins that are optionally isolated from a cellular orcell-free translation system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates certain specific compounds useful in the compositionsand methods of the invention, according to various embodiments.

FIG. 2A and FIG. 2B illustrate certain specific compounds useful in thecompositions and methods of the invention, according to variousembodiments.

FIG. 3 shows an image of a thin layer chromatography (TLC) platecaptured on an Ambis Imaging system set to detect the fluorescentemission from fluorescent species present on the TLC plate when exposedto ultraviolet light and collected through a filter excludingultraviolet light present on the imaging camera.

FIG. 4 illustrates the relative degree of labeling observed when acompound of the invention is incubated with a protein having anN-terminal cysteine residue versus an identical reaction where theprotein having an N-terminal Cys residue is replaced with an equalamount of a protein differing from the N-terminal Cys protein only inthat the second protein has an N-terminal alanine residue, both of whichcan be generated by cleavage with TEV protease, as described in Example5.

FIG. 5A, FIG. 5B, and FIG. 5C shows the fluorescent gel images andCoomassie stained gels prepared according to Example 5, illustratingthat a protein with an N-terminal cysteine can be prepared by cleaving afusion construct with TEV protease. The protein can be labeled at itsN-terminus with a cyanobenzothiazole reagent, which can then beselectively cleaved with a second protease in a subsequent step.

FIG. 6 illustrates peptide chain cleavage and Coomassie stained gels forpeptides exclusively labeled at the N-terminus, according to theprocedure of Example 5. The abbreviation CN-BT indicates acyanobenzothiazole derivative reagent as described herein; HT=HaloTag;GST=Glutathione-S-tranferase; TMR=tetramethylrhodamine; UC=uncut; andFXa=Factor Xa protease cut.

FIG. 7 illustrates peptide chain cleavage and Coomassie stained gels fornon-specifically labeled peptides (both internal cysteines andN-terminal cysteines), according to the procedure of Example 5. Theabbreviation CN-BT indicates a cyanobenzothiazole derivative reagent asdescribed herein; HT=HaloTag; GST=Glutathione-S-tranferase;TMR=tetramethylrhodamine; UC=uncut; and FXa=Factor Xa protease cut.

FIG. 8A and FIG. 8B illustrate the results of a label used to detectprotein:protein interactions as described in Example 6.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the following terms and expressions have the indicatedmeanings. It will be appreciated that the compounds of the presentinvention contain asymmetrically substituted carbon atoms, and may beisolated in optically active or racemic forms. It is well known in theart how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis from optically active starting materials.All chiral, diastereomeric, racemic forms and all geometric isomericforms of a structure are part of this invention.

Specific values listed below for radicals, substituents, and ranges, arefor illustration only; they do not exclude other defined values or othervalues within defined ranges for the radicals and substituents.

As used herein, the term “substituted” is intended to indicate that oneor more (e.g., 1, 2, 3, 4, or 5; in some embodiments 1, 2, or 3; and inother embodiments 1 or 2) hydrogens on a group is replaced with one ormore “substituents”, e.g., one or more of a selection of suitable groupknown to those of skill in the art, provided that the indicated atom'snormal valency is not exceeded, and that the substitution results in astable compound. Suitable substituents include, e.g., alkyl, alkenyl,alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl,heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino,alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl,acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy,carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl,arylsulfinyl, arylsulfonyl, heteroarylsulfinyl, heteroarylsulfonyl,heterocyclesulfinyl, heterocyclesulfonyl, phosphate, sulfate, hydroxylamine, hydroxyl (alkyl)amine, and cyano. Additionally, the suitableindicated groups can include, e.g., —X, —R, —O⁻, —OR, —SR, —S⁻, —NR₂,—NR₃, ═NR, —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO, —NO₂, ═N₂, —N₃,NC(═O)R, —C(═O)R, —C(═O)NRR—S(═O)₂O⁻, —S(═O)₂OH, —S(═O)₂R, —OS(═O)₂OR,—S(═O)₂NR, —S(═O)R, —OP(═O)O₂RR, —P(═O)O₂RR—P(═O)(O⁻)₂, —P(═O)(OH)₂,—C(═O)R, —C(═O)X, —C(S)R, —C(O)OR, —C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR,—C(O)NRR, —C(S)NRR, —C(NR)NRR, where each X is independently a halogen(“halo”): F, Cl, Br, or I; and each R is independently H, alkyl, aryl,heteroaryl, heterocycle, a protecting group or prodrug moiety. As wouldbe readily understood by one skilled in the art, when a substituent isketo (═O) or thioxo (═S), or the like, then two hydrogen atoms on thesubstituted atom are replaced. In some embodiments, one or more of thepreceding groups can be expressly excluded from an embodiment.

The terms “stable compound” and “stable structure” indicate a compoundthat is sufficiently robust to survive isolation to a useful degree ofpurity from a reaction mixture. Only stable compounds are claimed in thepresent invention, however, certain unstable compounds, for example,those that cannot easily be isolated, can be employed in the methodsdescribed herein.

One diastereomer may display superior properties or activity comparedwith another. When required, separation of the racemic material can beachieved by HPLC using a chiral column or by a resolution using aresolving agent such as camphonic chloride as described by Tucker etal., J. Med. Chem., 37: 2437 (1994). A chiral compound may also bedirectly synthesized using a chiral catalyst or a chiral ligand, e.g.Huffman et al., J. Org. Chem., 60: 1590 (1995).

As used herein, the term “alkyl” refers to a branched, unbranched, orcyclic hydrocarbon having, for example, from 1 to 20 carbon atoms, andoften 1 to about 12, 1 to about 6, or 1 to about 4 carbon atoms.Examples include, but are not limited to, methyl, ethyl, 1-propyl,2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl(t-butyl), 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl,3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl,3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl,3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl,3,3-dimethyl-2-butyl, hexyl, octyl, decyl, dodecyl, and the like. Thealkyl can be unsubstituted or substituted. The alkyl can also beoptionally partially or fully unsaturated. As such, the recitation of analkyl group includes both alkenyl and alkynyl groups. The alkyl can be amonovalent hydrocarbon radical, as described and exemplified above, orit can be a divalent hydrocarbon radical (i.e., alkylene).

The term “alkenyl” refers to a monoradical branched or unbranchedpartially unsaturated hydrocarbon chain (i.e. a carbon-carbon, sp²double bond). In one embodiment, an alkenyl group can have from 2 to 10carbon atoms, or 2 to 6 carbon atoms. In another embodiment, the alkenylgroup has from 2 to 4 carbon atoms. Examples include, but are notlimited to, ethylene or vinyl, allyl, cyclopentenyl, 5-hexenyl, and thelike. The alkenyl can be unsubstituted or substituted.

The term “alkynyl” refers to a monoradical branched or unbranchedhydrocarbon chain, having a point of complete unsaturation (i.e. acarbon-carbon, sp triple bond). In one embodiment, the alkynyl group canhave from 2 to 10 carbon atoms, or 2 to 6 carbon atoms. In anotherembodiment, the alkynyl group can have from 2 to 4 carbon atoms. Thisterm is exemplified by groups such as ethynyl, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 3-butynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl,1-octynyl, and the like. The alkynyl can be unsubstituted orsubstituted.

The term “cycloalkyl” refers to cyclic alkyl groups of from 3 to 10carbon atoms having a single cyclic ring or multiple condensed rings.Such cycloalkyl groups include, by way of example, single ringstructures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, andthe like, or multiple ring structures such as adamantanyl, and the like.The cycloalkyl can be unsubstituted or substituted. The cycloalkyl groupcan be monovalent or divalent, and can be optionally substituted asdescribed above for alkyl groups. The cycloalkyl group can optionallyinclude one or more cites of unsaturation, for example, the cycloalkylgroup can include one or more carbon-carbon double bonds, such as, forexample, cyclohexene, 1,3-cyclohexadiene, 1,4-cyclohexadiene, and thelike.

The term “alkoxy” refers to the group alkyl-O—, where alkyl is asdefined herein. In one embodiment, alkoxy groups include, e.g., methoxy,ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy,n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like. The alkoxy can beunsubstituted or substituted.

As used herein, “aryl” refers to an aromatic hydrocarbon group derivedfrom the removal of one hydrogen atom from a single carbon atom of aparent aromatic ring system. The radical can be at a saturated orunsaturated carbon atom of the parent ring system. The aryl group canhave from 6 to 20 carbon atoms. The aryl group can have a single ring(e.g., phenyl) or multiple condensed (fused) rings, wherein at least onering is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, oranthryl). Typical aryl groups include, but are not limited to, radicalsderived from benzene, naphthalene, anthracene, biphenyl, and the like.The aryl can be unsubstituted or optionally substituted, as describedabove for alkyl groups.

The term “halo” refers to fluoro, chloro, bromo, and iodo. Similarly,the term “halogen” refers to fluorine, chlorine, bromine, and iodine.

The term “haloalkyl” refers to alkyl as defined herein substituted by 1or more halo groups as defined herein, which may be the same ordifferent. In one embodiment, the haloalkyl can be substituted with 1,2, 3, 4, or 5 halo groups. In another embodiment, the haloalkyl can bysubstituted with 1, 2, or 3 halo groups. The term haloalkyl also includeperfluoro-alkyl groups. Representative haloalkyl groups include, by wayof example, trifluoromethyl, 3-fluorododecyl, 12,12,12-trifluorododecyl,2-bromooctyl, 3-bromo-6-chloroheptyl, 1H,1H-perfluorooctyl, and thelike. The haloalkyl can be optionally substituted as described above foralkyl groups.

The term “heteroaryl” is defined herein as a monocyclic, bicyclic, ortricyclic ring system containing one, two, or three aromatic rings andcontaining at least one nitrogen, oxygen, or sulfur atom in an aromaticring, and that can be unsubstituted or substituted, for example, withone or more, and in particular one to three, substituents, as describedabove in the definition of “substituted”. Typical heteroaryl groupscontain 2-20 carbon atoms in addition to the one or more hetoeroatoms.Examples of heteroaryl groups include, but are not limited to,2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, acridinyl, benzo[b]thienyl,benzothiazolyl, □-carbolinyl, carbazolyl, chromenyl, cinnolinyl,dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl,indazolyl, indolisinyl, indolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl,perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl,phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl,pyridyl, pyrimidinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl,quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl,tetrazolyl, and xanthenyl. In one embodiment the term “heteroaryl”denotes a monocyclic aromatic ring containing five or six ring atomscontaining carbon and 1, 2, 3, or 4 heteroatoms independently selectedfrom non-peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H,O, alkyl, aryl, or (C₁-C₆)(alkyl)aryl. In another embodiment heteroaryldenotes an ortho-fused bicyclic heterocycle of about eight to ten ringatoms derived therefrom, particularly a benz-derivative or one derivedby fusing a propylene, trimethylene, or tetramethylene diradicalthereto.

The term “heterocycle” refers to a saturated or partially unsaturatedring system, containing at least one heteroatom selected from the groupoxygen, nitrogen, and sulfur, and optionally substituted with one ormore groups as defined herein under the term “substituted”. Aheterocycle can be a monocyclic, bicyclic, or tricyclic group containingone or more heteroatoms. A heterocycle group also can contain an oxogroup (═O) or a thioxo (═S) group attached to the ring. Non-limitingexamples of heterocycle groups include 1,3-dihydrobenzofuran,1,3-dioxolane, 1,4-dioxane, 1,4-dithiane, 2H-pyran, 2-pyrazoline,4H-pyran, chromanyl, imidazolidinyl, imidazolinyl, indolinyl,isochromanyl, isoindolinyl, morpholine, piperazinyl, piperidine,piperidyl, pyrazolidine, pyrazolidinyl, pyrazolinyl, pyrrolidine,pyrroline, quinuclidine, and thiomorpholine.

The term “heterocycle” can include, by way of example and notlimitation, a monoradical of the heterocycles described in Paquette, LeoA.; Principles of Modern Heterocyclic Chemistry (W.A. Benjamin, NewYork, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; The Chemistryof Heterocyclic Compounds, A Series of Monographs” (John Wiley & Sons,New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and28; and J. Am. Chem. Soc., 82: 5566 (1960). In one embodiment,“heterocycle” includes a “carbocycle” as defined herein, wherein one ormore (e.g. 1, 2, 3, or 4) carbon atoms have been replaced with aheteroatom (e.g. O, N, or S).

Examples of heterocycles, by way of example and not limitation, include,dihydroxypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, piperidinyl, 4-piperidonyl,pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl,octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl,2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl,isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl,isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl,isoindolyl, 3H-indolyl, 1H-indazoly, purinyl, 4H-quinolizinyl,phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl,pteridinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl,pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl,phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl,pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl,quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl,benzisoxazolyl, oxindolyl, benzoxazolinyl, isatinoyl, andbis-tetrahydrofuranyl.

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Carbon bonded heterocycles include 2-pyridyl, 3-pyridyl,4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl,5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl,5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl,6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, and the like.

By way of example and not limitation, nitrogen bonded heterocycles canbe bonded at position 1 of an aziridine, azetidine, pyrrole,pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine,2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline,3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole,position 2 of a isoindole, or isoindoline, position 4 of a morpholine,and position 9 of a carbazole, or β-carboline. In one embodiment,nitrogen bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl,1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

The term “carbocycle” refers to a saturated, unsaturated or aromaticring having 3 to 8 carbon atoms as a monocycle, 7 to 12 carbon atoms asa bicycle, and up to about 30 carbon atoms as a polycycle. Monocycliccarbocycles typically have 3 to 6 ring atoms, still more typically 5 or6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g.,arranged as a bicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10ring atoms arranged as a bicyclo [5,6] or [6,6] system. Examples ofcarbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, phenyl, spiryland naphthyl. The carbocycle can be optionally substituted as describedabove for alkyl groups.

The term “alkanoyl” or “alkylcarbonyl” refers to —C(═O)R, wherein R isan alkyl group as previously defined.

The term “acyloxy” or “alkylcarboxy” refers to —O—C(═O)R, wherein R isan alkyl group as previously defined. Examples of acyloxy groupsinclude, but are not limited to, acetoxy, propanoyloxy, butanoyloxy, andpentanoyloxy. Any alkyl group as defined above can be used to form anacyloxy group.

The term “alkoxycarbonyl” refers to —C(═O)OR (or “COOT”), wherein R isan alkyl group as previously defined.

The term “amino” refers to —NH₂. The amino group can be optionallysubstituted as defined herein for the term “substituted”. The term“alkylamino” refers to —NR₂, wherein at least one R is alkyl and thesecond R is alkyl or hydrogen. The term “acylamino” refers toN(R)C(═O)R, wherein each R is independently hydrogen, alkyl, or aryl.

The term “amino acid,” includes a residue of a natural amino acid (e.g.Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well asunnatural amino acids (e.g. phosphoserine, phosphothreonine,phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid,octahydroindole-2-carboxylic acid, statine,1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine,ornithine, citruline, α-methyl-alanine, para-benzoylphenylalanine,phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). Theterm also comprises natural and unnatural amino acids bearing aconventional amino protecting group (e.g. acetyl or benzyloxycarbonyl),as well as natural and unnatural amino acids protected at the carboxyterminus (e.g. as a (C₁-C₆)alkyl, phenyl or benzyl ester or amide; or asan α-methylbenzyl amide). Other suitable amino and carboxy protectinggroups are known to those skilled in the art (See for example, Greene,T. W.; Wutz, P. G. M. Protecting Groups In Organic Synthesis, 2^(nd)edition, John Wiley & Sons, Inc., New York (1991) and references citedtherein).

The term “peptide” describes a sequence of 2 to 35 amino acids (e.g. asdefined hereinabove) or peptidyl residues. The sequence may be linear orcyclic. For example, a cyclic peptide can be prepared or may result fromthe formation of disulfide bridges between two cysteine residues in asequence. Preferably a peptide comprises 3 to 20, or 5 to 15 aminoacids. Peptide derivatives can be prepared as disclosed in U.S. Pat.Nos. 4,612,302; 4,853,371; and 4,684,620, or as described in theExamples herein below. Peptide sequences specifically recited herein arewritten with the amino terminus on the left and the carboxy terminus onthe right.

The term “saccharide” refers to a sugar or other carbohydrate,especially a simple sugar. The saccharide can be a C₆-polyhydroxycompound, typically C₆-pentahydroxy, and often a cyclic glycal. The termincludes the known simple sugars and their derivatives, as well aspolysaccharides with two or more monosaccaride residues. The saccharidecan include protecting groups on the hydroxyl groups, as described abovein the definition of amino acids. The hydroxyl groups of the saccharidecan be replaced with one or more halo or amino groups. Additionally, oneor more of the carbon atoms can be oxidized, for example to keto orcarboxyl groups.

The term “interrupted” indicates that another group is inserted betweentwo adjacent carbon atoms (and the hydrogen atoms to which they areattached (e.g., methyl (CH₃), methylene (CH₂) or methine (CH))) of aparticular carbon chain being referred to in the expression using theterm “interrupted”, provided that each of the indicated atoms' normalvalency is not exceeded, and that the interruption results in a stablecompound. Suitable groups that can interrupt a carbon chain include,e.g., with one or more non-peroxide oxy (—O—), thio (—S—), imino(—N(H)—), methylene dioxy (—OCH₂O—), carbonyl (—C(═O—), carboxy(—C(═O)O—), carbonyldioxy (—OC(═O)O—), carboxylato (—OC(═O—), imine(C═NH), sulfinyl (SO) and sulfonyl (SO₂). Alkyl groups can beinterrupted by one ore more (e.g., 1, 2, 3, 4, 5, or about 6) of theaforementioned suitable groups. The site of interruption can also bebetween a carbon atom of an alkyl group and a carbon atom to which thealkyl group is attached. In certain embodiments, one or more of theaforementioned groups are excluded from an embodiment.

As to any of the above groups, which contain one or more substituents,it is understood, of course, that such groups do not contain anysubstitution or substitution patterns that are sterically impracticaland/or synthetically non-feasible. In addition, the compounds of thisinvention include all stereochemical isomers arising from thesubstitution of these compounds. In certain embodiments, the compoundsof the invention do not include any compounds disclosed in U.S. Pat. No.5,424,440 (Klem et al.).

Selected substituents within the compounds described herein are presentto a recursive degree. In this context, “recursive substituent” meansthat a substituent may recite another instance of itself. Because of therecursive nature of such substituents, theoretically, a large number maybe present in any given claim. One of ordinary skill in the art ofmedicinal chemistry and organic chemistry understands that the totalnumber of such substituents is reasonably limited by the desiredproperties of the compound intended. Such properties include, by ofexample and not limitation, physical properties such as molecularweight, solubility or log P, application properties such as activityagainst the intended target, and practical properties such as ease ofsynthesis.

Recursive substituents are an intended aspect of the invention. One ofordinary skill in the art of medicinal and organic chemistry understandsthe versatility of such substituents. To the degree that recursivesubstituents are present in an claim of the invention, the total numberwill be determined as set forth above.

The term “linker” as used herein is an atom chain, typically a carbonchain, that covalently attaches two chemical groups together and mayinclude a substrate for an enzyme that may be cleaved by that enzyme oranother molecule, or may be photosensitive. The chain is optionallyinterrupted by one or more nitrogen atoms, oxygen atoms, carbonylgroups, (substituted) aromatic rings, or peptide bonds, and/or one ofthese groups may occur at one or both ends of the atom chain that formsthe linker. Many linkers are well known in the art, and can be used tolink a compound or formula described herein to another group, such as asolid support or resin. See for example, the linkers and solid supportsdescribed by Sewald and Jakubke in Peptides: Chemistry and Biology,Wiley-VCH, Weinheim (2002), pages 212-223; and by Dorwald in OrganicSynthesis on Solid Phase, Wiley-VCH, Weinheim (2002).

As used herein, a “fluorophore” includes a molecule that is capable ofabsorbing energy at a wavelength range and releasing energy at awavelength range other than the absorbance range. In certainembodiments, the fluorophore is a molecule that is capable of absorbingenergy at about 250 nm to about 900 nm, and can release energy at awavelength range of about 260 nm to about 910 nm. The term “excitationwavelength” refers to the range of wavelengths at which a fluorophoreabsorbs energy. The term “emission wavelength” refers to the range ofwavelengths that the fluorophore releases energy or fluoresces.

Fluorophores include but are not limited to fluoroscein, Texas Red,DAPI, PI, acridine orange, Alexa fluors, e.g., Alexa 350, Alexa 405 orAlexa 488, cyanine dyes such as Cy3, Cy5, and Cy7, coumarin, ethidiumbromide, fluorescein, BODIPY, rhodol, Rox, 5-carboxyfluorescein,6-carboxyfluorescein, an anthracene, 2-amino-4-methoxynapthalene, aphenalenone, an acridone, fluorinated xanthene derivatives, α-naphtol,β-napthol, 1-hydroxypyrene, coumarins, e.g., 7-amino-4-methylcoumarin(AMC) or 7-amino-4-trifluoromethylcoumarin (AFC), rhodamines, e.g.,tetramethylrhodamine, rhodamine-110, carboxyrhodamine, cresyl violet, orresorufin, as well as fluorophores disclosed in U.S. Pat. No. 6,420,130(Makings, et al.), the disclosure of which is incorporated by referenceherein. Fluorophores include cyanine dyes, such as compounds of theformula Ar—[CH═CH]_(n)—[CH═]_(m)Ar, wherein Ar is an aryl or heteroarylgroup; n is 1, 2, 3, or 4; m is 0 or 1; and wherein each Ar includes aquaternary nitrogen or a nitrogen capable of being quaternized throughresonance. Examples of such aryl or heteroaryl groups includedimethyl-aminophenyl, imidazole, pyridine, pyrrole, quinoline, thiazole,and indole, each optionally substituted. The fluorophore can be acompound that is inherently fluorescent or demonstrates a change influorescence upon binding to a biological compound, i.e. it can befluorogenic, or its intensity can be diminished by quenching.Fluorophores may contain substitutents that alter the solubility,spectral properties or physical properties of the fluorophore. Variousfluorophores are known to those skilled in the art and also include, butare not limited to benzofurans, quinolines, quinazolinones, indoles,benzazoles, borapolyazaindacene, and xanthenes including fluoroscein,rhodamine, and rhodol, as well as other fluorophores described inRichard P. Haugland's The Handbook, A Guide to Fluorescent Probes andLabeling Technologies (10^(th) edition, 2005), which describes numerousfluorophones available from Invitrogen Molecular Probes.

A “fluorogenic assay” or “fluorogenic reaction” includes a reaction inwhich a product of a reaction is fluorescent. A “fluorogenic assayreagent” may include a substrate, as well as a cofactor(s) or othermolecule(s) such as a protein, e.g., an enzyme, for a fluorogenicreaction.

The term “solid support” refers to a support that can be isolated from areaction mixture in solid form, such as a silicate or polymer particle.Solid supports include various particles and surfaces, such as beads,microtiter plates, eppendorf tubes, and slides. The surface can be apolymer such as sepharose, cellulose, alginate, polystyrene or otherplastics, and/or other surfaces including membranes and glass. Manycommon solid supports are described by Sewald and Jakubke in Peptides:Chemistry and Biology, Wiley-VCH, Weinheim (2002), pages 212-223; and byDorwald in Organic Synthesis on Solid Phase, Wiley-VCH, Weinheim (2002).The cyanobenzothiazoles can be linked to the solid support eithernon-specifically (via adsorption to the surface) or specifically (viacapture by an antibody specific to the cyanobenzothiazole, as in a“sandwich” ELISA). A detection antibody on the surface of the solidsupport can be covalently linked to an enzyme, or can itself be detectedby a secondary antibody linked to an enzyme through bioconjugation. Forexamples of ELISA “sandwich” test procedures, see Schuurs and vanWeemen, J. Immunoassay 1980; 1:229-49.

The term “reporter moiety” refers to a portion of a molecule that can bedetected in a biological or non-biological mixture (e.g. a fluorophore,chromophore, or radioactive element). The reporter moiety can be amolecule that is capable of functioning as a member of an energytransfer pair wherein the reporter molecule retains its nativeproperties (e.g., spectral properties, conformation, and/or activity)when attached to a ligand analog and is used in the methods disclosedherein. The reporter moiety can be a reporter molecule linked to acyanobenzothiazole. Examples of reporter molecules include but are notlimited to nucleic acids, borapolyazaindacenes, coumarins, xanthenes,cyanines, and luminescent molecules, including dyes, fluorescentproteins, chromophores, and chemiluminescent compounds that are capableof producing a detectable signal upon appropriate activation. The term“dye” refers to a compound that emits light to produce an observabledetectable signal. “Dye” includes phosphorescent, fluorescent, andnonfluorescent compounds that include without limitation pigments,fluorophores, chemiluminescent compounds, luminescent compounds, andchromophores. The term “chromophore” refers to a label that emits and/orreflects light in the visible spectra that can be observed without theaid of instrumentation.

The terms “affinity moiety”, “affinity label”, and/or “affinitymolecule” refer to a portion of a molecule that can effectively bindnoncovalently or covalently to a molecule, biomolecule, or material ofinterest (e.g. biotin, HisTag, or chitin). An affinity moiety can be amolecule containing acceptor groups. Thus, a cyanobenzothiazolederivative that includes an affinity moiety can be used to facilitatethe identification and separation of labeled molecules or complexesbecause of the selective interaction of the affinity moiety with anothermolecule, e.g., a molecule that will bind to the affinity moiety, thatmay be biological or non-biological in origin.

The term “quencher” or “quenching moiety” refers to a molecule orportion of a molecule that is a strong photon absorber, isnon-fluorescent or is essentially non-fluorescent, and effectivelyquenches fluorescence of other molecules in its vicinity. The quenchingmoiety can be a moiety that is capable of absorbing energy from anenergy donor that is not re-emitted (non-fluorescent), or is re-emittedat a detectably different wavelength from the energy emitted by thedonor molecule. In this respect, in certain embodiments quenchers may beessentially non-fluorescent or fluorescent. Some examples of quenchermoieties that can be linked to a cyanobenzothiazole include xanthene,xanthene derivatives, cyanine, cyanine derivatives,dimethylaminoazosulfonic acid (DABSYL), and dimethylaminoazo-carboxylicacid (DABCYL). Numerous quenching moieties are well known in the artincluding xanthenes, cyanines, and other compounds disclosed in RichardP. Haugland's The Handbook, A Guide to Fluorescent Probes and LabelingTechnologies (10^(th) edition, 2005). Quenchers and quenching offluorescence is further described by J. Lakowicz in Principles ofFluorescence Spectroscopy, 2^(nd) Ed., New York: Kluwer Academic/Plenum(1999); see in particular, Chapter 8 (“Quenching of Fluorescence”),pages 237-264; and the 3^(rd) Ed., New York: Springer Science (2006),Chapter 8, pages 278-327, and references cited therein. In certainembodiments, a quencher can be a chromophoric molecule or part of acompound, capable of reducing the emission from a fluorescent donor whenattached to the donor. Quenching may occur by any of several mechanismsincluding fluorescence resonance energy transfer, photoinduced electrontransfer, paramagnetic enhancement of intersystem crossing, Dexterexchange coupling, and exciton coupling such as the formation of darkcomplexes.

The term “acceptor” refers to a quencher that operates via energytransfer. Acceptors may re-emit the transferred energy as fluorescenceand are “acceptor fluorescent moieties”. Examples of acceptors includecoumarins and related fluorophores, xanthenes such as fluoresceins,rhodols, and rhodamines, resorufins, cyanines,difluoroboradiazaindacenes, and phthalocyanines. Other chemical classesof acceptors generally do not re-emit the transferred energy as light.Examples include some indigos, benzoquinones, anthraquinones, azocompounds, nitro compounds, indoanilines, and di- and triphenylmethanes.

The term “photocrosslinking moiety” refers to a portion of a moleculewhich upon photoexcitation can covalently bond to another molecule,biomolecule, or material of interest. For example, a compound thatincludes a photocrosslinking moiety can be used to crosslink a proteinupon photoexcitation.

The term “enzyme of interest” refers to any enzyme that can be labeledusing the methods of the invention or other methods known in the art.Enzymes of interest include, for example, kinases, phosphatases,peroxidases, sulfatases, peptidases, glycosidases, proteases, forexample, proteases involved in apoptosis, hydrolases, oxidoreductases,lyases, transferases, isomerases, ligases, protein kinases, proteinphosphatases, esterases, isomerases, glycosylases, synthetases,dehydrogenases, oxidases, reductases, methylases and the like. Furtherenzymes of interest include those involved in making or hydrolyzingesters, both organic and inorganic, glycosylating, and hydrolyzing amidebonds. In any class, there may be further subdivisions, as in thekinases, where the kinase may be specific for phosphorylation of serine,threonine and/or tyrosine residues in peptides and proteins.

Other enzymes of interest include any protein that exhibits enzymaticactivity, e.g., lipases, phospholipases, sulphatases, ureases,peptidases, proteases and esterases, including acid phosphatases,glucosidases, glucuronidases, galactosidases, carboxylesterases, andluciferases. In one embodiment, the enzyme is a hydrolytic enzyme.Examples of hydrolytic enzymes include alkaline and acid phosphatases,esterases, decarboxylases, phospholipase D, P-xylosidase,β-D-fucosidase, thioglucosidase, β-D-galactosidase, α-D-galactosidase,α-D-glucosidase, β-D-glucosidase, β-D-glucuronidase, α-D-mannosidase,β-D-mannosidase, β-D-fructofuranosidase, and β-D-glucosiduronase.

Further enzymes of interest are hydrolases, including but are notlimited to, enzymes acting on ester bonds such as carboxylic esterhydrolases, thiolester hydrolases, phosphoric monoester hydrolases,phosphoric diester hydrolases, triphosphoric monoester hydrolases,sulfuric ester hydrolases, diphosphoric monoester hydrolases, phosphorictriester hydrolases, exodeoxyribonucleases producing5′-phosphomonoesters, exoribonucleases producing 5′-phosphomonoesters,exoribonucleases producing 3′-phosphomonoesters, exonucleases activewith either ribo- or deoxyribonucleic acid, exonucleases active witheither ribo- or deoxyribonucleic acid, endodeoxyribonucleases producing5′-phosphomonoesters, endodeoxyribonucleases producing other than5′-phosphomonoesters, site-specific endodeoxyribonucleases specific foraltered bases, endoribonucleases producing 5′-phosphomonoesters,endoribonucleases producing other than 5′-phosphomonoesters,endoribonucleases active with either ribo- or deoxyribonucleic,endoribonucleases active with either ribo- or deoxyribonucleicglycosylases; glycosidases, e.g., enzymes hydrolyzing O- and S-glycosylcompounds, and those hydrolyzing N-glycosyl compounds; acting on etherbonds such as trialkylsulfonium hydrolases or ether hydrolases; enzymesacting on peptide bonds (peptide hydrolases) such as aminopeptidases,dipeptidases, dipeptidyl-peptidases and tripeptidyl-peptidases,peptidyl-dipeptidases, serine-type carboxypeptidases,metallocarboxypeptidases, cysteine-type carboxypeptidases, omegapeptidases, serine endopeptidases, cysteine endopeptidases, asparticendopeptidases, metalloendopeptidases, threonine endopeptidases, andendopeptidases of unknown catalytic mechanism; enzymes acting oncarbon-nitrogen bonds, other than peptide bonds, such as those in linearamides, in cyclic amides, in linear amidines, in cyclic amidines, innitriles, or other compounds; enzymes acting on acid anhydrides such asthose in phosphorous-containing anhydrides and in sulfonyl-containinganhydrides; enzymes acting on acid anhydrides (catalyzing transmembranemovement); enzymes acting on acid anhydrides or involved in cellular andsubcellular movement; enzymes acting on carbon-carbon bonds (e.g., inketonic substances); enzymes acting on halide bonds (e.g., in C-halidecompounds), enzymes acting on phosphorus-nitrogen bonds; enzymes actingon sulfur-nitrogen bonds; enzymes acting on carbon-phosphorus bonds; andenzymes acting on sulfur-sulfur bonds.

The term “poly-histidine tract” or “His tag” refers to a moleculecomprising two to ten histidine residues, e.g., a poly-histidine tractof five to ten residues. A poly-histidine tract allows the affinitypurification of a covalently linked molecule on an immobilized metal(e.g., nickel, zinc, cobalt or copper) chelate column or through aninteraction with another molecule (e.g., an antibody reactive with theHis tag).

Linkers

A linker strategy may be employed to link a reporter moiety, e.g., afluorophore, an affinity moiety, or another labeling group such as abiotin, a resin, a carbohydrate, an oligopeptide, a dye, or a drugmoiety, to a cyanobenzothiazole, yielding a cyanobenzothiazolederivative capable of reacting with a protein that has an N-terminalcysteine. The use of linkers or “linking groups” is well known in theart.

A linking group can be an alkyl or alkoxy chain, such as a (C₁-C₆)alkylor a (C₁-C₆)alkoxy group. The chain can have one or more electronwithdrawing group substituents R, such as an aldehyde, acetyl,sulfoxide, sulfone, nitro, cyano group, or a combination thereof.Certain linkers and methods for preparing covalent linkages aredescribed in, for example, U.S. Pat. No. 7,282,339 (Beechem et al.); inPeptides: Chemistry and Biology by Sewald and Jakubke, Wiley-VCH,Weinheim (2002), pages 212-223; and in Organic Synthesis on Solid Phaseby Dorwald, Wiley-VCH, Weinheim (2002); which are incorporated herein byreference.

In certain embodiments, the linking group can be a divalent radical ofthe formula W-A wherein A is (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, (C₃-C₈)cycloalkyl, or (C₆-C₁₀)aryl; W is —N(R)C(═O)—,—C(═O)N(R)—, —OC(═O)—, —C(═O)O—, —O—, —S—, —S(O)—, —S(O)₂—, —N(R)—,—C(═O)—, or a direct bond; each R is independently H, (C₁-C₆)alkyl, or aprotecting group; and the linker group links together two othermolecular moieties, for example, a cyanobenzothiazole moiety and a groupX as defined above, such as a reporter moiety.

Immobilization

The invention includes immobilized derivatives of cyanobenzothiazole andmethods to immobilize the cyanobenzothiazole derivative. Someimmobilized cyanobenzothiazoles include those that are linked to solidsupports at a position on the benzo ring, for example, at the6′-position. Other immobilized cyanobenzothiazoles include those thatare bound to the surface of a solid support by bioactive groups (e.g.,biotin, avidin, streptavidin, or derivatives thereof), non-covalentbinding interactions such as by antibodies, or antigens, or His-tag to anickel column. Still other immobilized cyanobenzothiazole can beprepared by reacting the cyanobenzothiazole with a terminalCys-containing protein of interest followed by binding with a solidsupport that has an appropriate antibody on the solid support surfacethat will bind the protein of interest.

Labeling Methods

The present invention comprises methods for the labeling of proteins anddetection and/or isolation of labeled proteins from a cellular orcell-free translation system. The isolated protein may be used directlyor further purified and/or manipulated. In one embodiment, the methodsmay be employed with defined proteins, e.g., a population of definedproteins, or employed with a set of proteins which include one or moreundefined proteins, such as those obtained from cells or an expressionlibrary.

The one or more proteins may be from a sample that includes eukaryoticcells, e.g., yeast, avian, plant, insect or mammalian cells, includingbut not limited to human, simian, murine, canine, bovine, equine,feline, ovine, caprine or swine cells, or prokaryotic cells, or cellsfrom two or more different organisms, or cell lysates or supernatantsthereof.

The methods disclosed herein comprise labeling proteins with Cys attheir N-terminus with any detectable molecule or a molecule that iscapable of being detected. The Cys may be added to the protein viarecombinant techniques, e.g., exposing a Cys such as one in a fusionprotein by intein-mediated splicing or insertion of an appropriateprotease site, the Cys may be naturally occurring at position 2 in aprotein susceptible to an N-terminal aminopeptidase, or may be added bysynthesis various synthesis techniques, e.g., via peptide ligation, orreverse proteolysis (see for example, Wehofsky, J. Amer. Chem. Soc.125:6126 (2003) and Chang, P.N.A.S. 91:12, 544 (1994)). For example, apreparation of proteins at least one of which has a N-terminal Cys isreacted with a derivative of cyanobenzothiazole having a fluorophore toform at least one protein that comprises the fluorophore covalentlylinked to its N-terminus. A linker group may be employed to facilitatelinking the reporter or affinity moiety to the cyanobenzothiazole.

A. Exemplary Moieties for Labeling

Labels include molecules (moieties) that are detectable or capable ofdetection and, in one embodiment, moieties useful in the compounds andmethods described herein are molecules that are capable of beingcovalently linked to the amino group of Cys via a derivative ofcyanobenzothiazole. Moieties useful in the compounds and methods haveone or more properties that facilitate detection and optionally thequantification and/or isolation of proteins comprising the moiety. Onephysical property is a characteristic electromagnetic spectral propertysuch as emission or absorbance, magnetism, electron spin resonance,electrical capacitance, dielectric constant or electrical conductivity.In certain embodiments, the moieties may be ferromagnetic, paramagnetic,diamagnetic, luminescent, electrochemi-luminescent, fluorescent,phosphorescent, chromatic, antigenic or have a distinctive mass.

Relevant moieties include, but are not limited to, a nucleic acidmolecule, i.e., DNA or RNA, e.g., an oligonucleotide or nucleotide, suchas one having nucleotide analogs, DNA which is capable of binding aprotein, single or double stranded DNA corresponding to a gene ofinterest, RNA corresponding to a gene of interest, mRNA which lacks astop codon, an aminoacylated initiator tRNA, an aminoacylated ambersuppressor tRNA, or double stranded RNA for RNAi, a protein, e.g., aluminescent protein, a peptide, a peptide nucleic acid, an epitoperecognized by a ligand, e.g., biotin or streptavidin, a hapten, an aminoacid, a lipid, a lipid bilayer, a solid support, a fluorophore, achromophore, a reporter molecule, a radionuclide, such as a radioisotopefor use in, for instance, radioactive measurements or a stable isotope,an electron opaque molecule, an X-ray contrast reagent, a MRI or X raycontrast agent, e.g., barium, iodine, manganese, gadolinium (III) oriron-oxide particles, and the like.

In one embodiment, the moiety is a glycoprotein, polysaccharide, tripletsensitizer, e.g., CALI, drug, toxin, lipid, biotin, or solid support,such as self-assembled monolayers, is electron opaque, is a chromophore,a nanoparticle, an enzyme, a substrate for an enzyme, an inhibitor of anenzyme, for instance, a suicide substrate, a cofactor, e.g., NADP, acoenzyme, a succinimidyl ester or aldehyde, glutathione, NTA, biotin,cAMP, phosphatidylinositol, a ligand for cAMP, a metal, a nitroxide ornitrone for use as a spin trap (detected by electron spin resonance(ESR), a metal chelator, e.g., for use as a contrast agent, in timeresolved fluorescence or to capture metals, a photocaged compound, e.g.,where irradiation liberates the caged compound such as a fluorophore, anintercalator, e.g., such as psoralen or another intercalator useful tobind DNA or as a photoactivatable molecule, a triphosphate or aphosphoramidite, e.g., to allow for incorporation of the substrate intoDNA or RNA, an antibody, a heterobifunctional cross-linker such as oneuseful to conjugate proteins or other molecules, cross-linkers includingbut not limited to hydrazide, aryl azide, maleimide,iodoacetamide/bromoacetamide, N-hydroxysuccinimidyl ester, mixeddisulfide such as pyridyl disulfide, glyoxal/phenylglyoxal, vinylsulfone/vinyl sulfonamide, acrylamide, boronic ester, hydroxamic acid,imidate ester, isocyanate/isothiocyanate, orchlorotriazine/dichlorotriazine, a glycoprotein, a polysaccharide,lipids including lipid bilayers; or is a solid support, e.g., asepharose or cellulose bead, a membrane, glass, e.g., glass slides,cellulose, alginate, plastic or other synthetically prepared polymer,e.g., an eppendorf tube or a well of a multi-well plate, self assembledmonolayers, a surface plasmon resonance chip, or a solid support with anelectron conducting surface, and includes a drug, an aminoacylated tRNAsuch as an aminoacylated initiator tRNA or an aminoacylated ambersuppressor tRNA, a molecule that binds Ca²⁺, a molecule that binds K⁺, amolecule that binds Na⁺, a molecule that is pH sensitive, aradionuclide, a molecule that is electron opaque, a molecule thatfluoresces in the presence of NO or is sensitive to a reactive oxygen, anonprotein substrate for an enzyme, an inhibitor of an enzyme, either areversible or irreversible inhibitor, a chelating agent, a cross-linkinggroup, for example, a succinimidyl ester or aldehyde, glutathione,biotin or other avidin binding molecule, avidin, streptavidin,phosphatidylinositol, heme, a ligand for cAMP, a metal, NTA, and, in oneembodiment, includes one or more dyes, e.g., a xanthene dye, a calciumsensitive dye, e.g.,1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)-phenoxy]-2-(2′-amino-5′-methylphenoxy)ethane-N,N,N′,N′-tetraaceticacid (Fluo-3), a sodium sensitive dye, e.g., 1,3-benzenedicarboxylicacid,4,4′-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-6,2-benzofurandiyl)]bis(PBFI), a NO sensitive dye, e.g.,4-amino-5-methylamino-2′,7′-difluorescein, or other fluorophore. In oneembodiment, the moiety is not a radionuclide. In another embodiment, themoiety is a radionuclide, e.g., ³H, ¹⁴C, ³⁵S, ¹²⁵I, ¹³¹I, including amolecule useful in diagnostic methods.

Exemplary moieties include haptens, e.g., molecules useful to enhanceimmunogenicity such as keyhole limpet hemacyanin (KLH), cleavablemoieties, for instance, photocleavable biotin, and fluorescent moieties,e.g., N-hydroxy-succinimide (NHS) modified coumarin and succinimide orsulfonosuccinimide modified BODIPY (which can be detected by UV and/orvisible excited fluorescence detection), rhodamine, e.g., R110, rhodols,CRG6, Texas Methyl Red (carboxytetramethylrhodamine),5-carboxy-X-rhodamine, or fluoroscein, coumarin derivatives, e.g.,7-aminocoumarin, and 7-hydroxycoumarin, 2-amino-4-methoxynapthalene,1-hydroxypyrene, resorufin, phenalenones or benzphenalenones (U.S. Pat.No. 4,812,409), acridinones (U.S. Pat. No. 4,810,636), anthracenes, andderivatives of α- and β-napthol, fluorinated xanthene derivativesincluding fluorinated fluoresceins and rhodols (e.g., U.S. Pat. No.6,162,931), bioluminescent molecules, e.g., luciferin, coelenterazine,luciferase, chemiluminescent molecules, e.g., stabilized dioxetanes, andelectrochemi-luminescent molecules.

Examples of affinity moieties include molecules such as immunogenicmolecules, e.g., epitopes of proteins, peptides, carbohydrates orlipids, i.e., any molecule which is useful to prepare antibodiesspecific for that molecule; biotin, avidin, streptavidin, andderivatives thereof; metal binding molecules; and fragments andcombinations of these molecules. Exemplary affinity molecules includeHis5 (HHHHH) (SEQ ID NO:1), HisX6 (HHHHHH) (SEQ ID NO:2), C-myc(EQKLISEEDL) (SEQ ID NO:3), Flag (DYKDDDDK) (SEQ ID NO:4), SteptTag(WSHPQFEK) (SEQ ID NO:5), HA Tag (YPYDVPDYA) (SEQ ID NO:6), thioredoxin,cellulose binding domain, chitin binding domain, S-peptide, T7 peptide,calmodulin binding peptide, C-end RNA tag, metal binding domains, metalbinding reactive groups, amino acid reactive groups, inteins, biotin,streptavidin, and maltose binding protein.

For example, the presence of the biotin at the N-terminus of proteinspermits selective binding of those proteins to avidin molecules, e.g.,avidin molecules coated onto a surface, e.g., beads, microwells,nitrocellulose and the like. Suitable surfaces include resins forchromatographic separation, plastics such as tissue culture surfaces forbinding plates, microtiter dishes and beads, ceramics and glasses,particles including magnetic particles, polymers and other matrices. Thetreated surface is washed with, for example, phosphate buffered saline(PBS), to remove non-nascent proteins and other translation reagents andthe nascent proteins isolated. In some case these materials may be partof biomolecular sensing devices such as optical fibers, chemfets, andplasmon detectors.

Another example of an affinity molecule is dansyllysine. Antibodies thatinteract with the dansyl ring system are commercially available (SigmaChemical; St. Louis, Mo.) or can be prepared using known protocols suchas those described in Antibodies: A Laboratory Manual (Harlow and Lane,1988). For example, the anti-dansyl antibody is immobilized onto thepacking material of a chromatographic column. This method, affinitycolumn chromatography, accomplishes separation by causing the complexbetween an immobilized antibody and a substrate to be retained on thecolumn (for example, a benzothiazole derivative linked to an affinitymoiety) due to its interaction with the immobilized antibody, whileother molecules pass through the column. The complex may then bereleased by disrupting the antibody-antigen interaction. Specificchromatographic column materials such as ion-exchange or affinitySepharose, Sephacryl, Sephadex and other chromatography resins arecommercially available (Sigma Chemical; St. Louis, Mo.; PharmaciaBiotech; Piscataway, N.J.). Dansyllysine may conveniently be detectedbecause of its fluorescent properties.

When employing an antibody as an acceptor molecule, separation can alsobe performed through other biochemical separation methods such asimmunoprecipitation and immobilization of antibodies on filters or othersurfaces such as beads, plates or resins. Beads are oftentimes separatedfrom the mixture using magnetic fields.

Another class of moieties includes molecules detectable usingelectromagnetic radiation and includes but is not limited to xanthenefluorophores, dansyl fluorophores, coumarins and coumarin derivatives,fluorescent acridinium moieties, benzopyrene based fluorophores, as wellas 7-nitrobenz-2-oxa-1,3-diazole, and3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diamino-propionic acid.Preferably, the fluorescent molecule has a high quantum yield offluorescence at a wavelength different from native amino acids and morepreferably has high quantum yield of fluorescence that can be excited inthe visible, or in both the UV and visible, portion of the spectrum.Upon excitation at a preselected wavelength, the molecule is detectableat low concentrations either visually or using conventional fluorescencedetection methods. Electrochemiluminescent molecules such as rutheniumchelates and its derivatives or nitroxide amino acids and theirderivatives are detectable at femtomolar ranges and below.

In one embodiment, an optionally detectable moiety includes one of:

wherein R₁ is, for example, (C₁-C₈)alkyl, optionally substituted withone or more substituents.

Methods that may be employed to detect and/or isolate moiety labeledproteins include chromatographic techniques including gel filtration,fast-pressure or high-pressure liquid chromatography, reverse-phasechromatography, affinity chromatography, ion exchange chromatography,electrophoresis, capillary electrophoresis and isoelectric focusing.Other methods of separation are also useful for detection and subsequentisolation, for example, electrophoresis, isoelectric focusing and massspectrometry.

Separation can also be performed through other biochemical separationmethods such as immunoprecipitation and immobilization of antibodies onfilters or other surfaces such as beads, plates or resins. For example,protein may be isolated by coating paramagnetic beads with aprotein-specific antibody. Beads are separated from the proteintranslation extract using magnetic fields.

Many devices designed to detect proteins are based on the interaction ofa target protein with a specific acceptor molecule, for instance, animmobilized acceptor molecule. Such devices can also be used to detectproteins once they contain affinity moieties such as biodetectors basedon sensing changes in surface plasmons, light scattering and electronicproperties of materials that are altered due to the interaction of thetarget molecule with the immobilized acceptor group.

B. Quenching Excess Labeling Reagent

In one embodiment, the fluorescence of excess un-reacted label (e.g.,compound 3028) may be quenched by addition of a quenching reagent to thelabeling reaction. The quenching reagent can be, for example, anybeta-mercaptoethylamine conjugated to a known fluorescent quencher. Onepossible example of such a quenching reagent is compound 3191.

The beta-mercaptoethylamine can react with the cyanobenzothiazole moietyof any unreacted labeling reagent, thereby conjugating the quencher tothe fluorophore of the labeling reagent. A quenching reagent of thissort would not react with or quench any labels that have previously beenconjugated to proteins.Moieties Detectable Using Electromagnetic Radiation

Moieties detectable using electromagnetic radiation include but are notlimited to dansyl fluorophores, coumarins and coumarin derivatives,fluorescent acridinium moieties, benzopyrene based fluorophores, as wellas 7-nitrobenz-2-oxa-1,3-diazole, and3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diamino-propionic acid. Inone embodiment, the fluorescent moiety has a high quantum yield offluorescence at a wavelength different from native amino acids and hashigh quantum yield of fluorescence which can be excited in either the UVor visible portion of the spectrum, or both the UV and visible portionsof the spectrum. Upon excitation at a preselected wavelength, the moietyis detectable at low concentrations either visually or usingconventional fluorescence detection methods. Electrochemiluminescentlabels such as ruthenium chelates and its derivatives or nitroxide aminoacids and their derivatives are detectable at the femtomolar ranges andbelow.

In addition to fluorescent moieties, a variety of moieties with physicalproperties based on the interaction and response of the moiety toelectromagnetic fields and radiation can be used to detect proteinproduction. These properties include absorption in the UV, visible andinfrared regions of the electromagnetic spectrum, presence ofchromophores which are Raman active, and can be further enhanced byresonance Raman spectroscopy, electron spin resonance activity andnuclear magnetic resonances and molecular mass, e.g., via a massspectrometer. These electromagnetic spectroscopic properties of themoiety are preferably not possessed by native amino acids or are readilydistinguishable from the properties of native amino acids.

Fluorescent and other moieties with detectable electromagnetic spectralproperties can be detected by various instruments, such as spectrometersor fluorometers and the like, and distinguished from the electromagneticspectral properties of native amino acids. Spectrometers are includefluorescence, Raman, absorption, electron spin resonance, visible,infrared and ultraviolet spectrometers. Other moieties, such as moietieswith distinct electrical properties can be detected by an apparatus suchas an ammeter, voltmeter or other spectrometer. Physical properties ofmoieties which relate to the distinctive interaction of the label withan electromagnetic field is readily detectable using instruments such asfluorescence, Raman, absorption, or electron spin resonancespectrometers. Moieties may also undergo a chemical, biochemical,electrochemical or photochemical reaction such as a color change inresponse to external forces or agents such as an electromagnetic fieldor reactant molecules which allows its detection.

Regardless of which class of fluorescent compounds is used, detectionmay involve physical separation of the proteins from other biomoleculespresent in the cellular or cell-free protein system. Protein separationcan be performed using, for example, gel electrophoresis or columnchromatography. Detection of a protein containing a fluorophore by gelelectrophoresis can be accomplished using conventional fluorescencedetection methods. After protein synthesis in a cell-free system, thereaction mixture, which contains all of the biomolecules necessary forprotein synthesis as well as proteins, is loaded onto a gel which may becomposed of polyacrylamide or agarose. Subsequent to loading thereaction mixture, a voltage is applied which spatially separates theproteins on the gel in the direction of the applied electric field. Theproteins separate and appear as a set of discrete or overlapping bandswhich can be visualized using a pre- or post-gel staining technique suchas Coomasie blue staining. The migration of the protein band on the gelis a function of the molecular weight of the protein with increasingdistance from the loading position being a function of decreasingmolecular weight. Bands on the gel which contain N-terminal Cys labeledproteins will exhibit fluorescence when excited at a suitablewavelength. These bands can be detected visually, photographically orspectroscopically and, if desired, the proteins purified from gelsections.

The molecular weight and quantity of the protein can be determined bycomparison of its band position on the gel with a set of bands ofproteins of predetermined molecular weight which are labeled, e.g.,fluorescently labeled. For example, a protein of molecular weight 25,000could be determined because of its relative position on the gel relativeto a calibration gel containing the commercially available standardmarker proteins of known quantities and with known molecular weights(bovine serum albumin, 66 kD; porcine heart fumarase, 48.5 kD; carbonicanhydrase, 29 kD, β-lactoglobulin, 18.4 kD; α-lactoglobulin, 14.2 kD;Sigma Chemical; St. Louis, Mo.). Calibration proteins may contain asimilar moiety for convenient detection using the same method as themoiety bearing the protein. This can be accomplished in many cases bydirectly reacting the calibration proteins with a molecule similar oridentical to the moiety. Thus, a calibration protein (protein marker),such as one or more selected based on pI or molecular weight, may belabeled by the method of the invention, e.g., using a fluorescein,rhodamine, BODIPY or infrared type moiety, and optionally isolated.

For example, the calibration proteins can be modified with dansylchloride or with a NHS modified BODIPY FL so as to obtain theirfluorescent counterparts. These fluorescent proteins can be analyzedusing PAGE. Combined detection of these fluorescent calibration proteinsalong with that of sample proteins which contain a fluorescent moietycan accurately determine both the molecular weight and quantity of theprotein synthesized. If necessary, the amounts of moiety within eachcalibration and protein can be determined to provide an accuratequantitation. Proteins with predetermined levels of a fluorescent moietycan be used advantageously to provide for quantitation of the moietybearing sample protein.

Other methods of protein separation are useful for detection andsubsequent isolation and purification of sample proteins containingmoieties detectable with electromagnetic radiation including capillaryelectrophoresis, isoelectric focusing, low pressure chromatography andhigh-performance or fast-pressure liquid chromatography. In these cases,the individual proteins are separated into fractions which can beindividually analyzed by fluorescent detectors at the emissionwavelengths of the moieties. Alternatively, on-line fluorescencedetection can be used to detect proteins as they emerge from the columnfractionation system. A graph of fluorescence as a function of retentiontime provides information on both the quantity and purity of proteinsproduced.

Uses and Exemplary Detection Methods for Labeled Proteins

The moiety containing proteins prepared by the methods of the inventionare useful for any purpose including but not limited to detect theamount or presence of a particular protein, to isolate a protein, tofacilitate high or low throughput screening, to detect protein-protein,protein-DNA or other protein-based interactions (e.g., using proteinmicroarrays in which the moiety is used to bind proteins to the array orto detect bound proteins), to enhance the immunogenecity of a protein(for example, the N-terminus of one or more proteins may be labeled witha hapten to enhance the production of antibodies to the protein, as wellas to facilitate antigen purification prior to immunization), to targeta protein to a particular cellular or subcellular location (e.g., alabel which is a protein localization domain may target the protein tothe nucleus, chloroplast or mitochondria, or to specific cells, e.g.,via a liver specific antibody), to provide site-specific orientation ofa protein, for example, a ligand for the moiety is attached to asemi-solid or solid surface or to a linker of any length, e.g., anorganic linker like polyethylene glycol (“PEG”), attached to asemi-solid or solid surface, such as glass, to prepare a chimericprotein comprising a reporter label, e.g., luciferase, and a protein ofinterest, e.g., CYP450, to prepare protein markers, or to map peptides,antigenic epitopes and binding sites on a protein.

Moreover, the labeled proteins find use in protein display technologiesand directed evolution. Ribosome, nucleic acid-protein fusion and phagedisplay technologies are widely being used to study protein-proteininteractions and directed evolution. In ribosome related displaytechnologies, an in vitro lysate expression system is employed for theproduction of mRNA-protein-ribosome complexes ormRNA-protein/cDNA-protein/DNA-protein fusion products (see publishedU.S. Application No. 2001/0046680, and U.S. Pat. Nos. 6,194,550;6,207,446; and 5,922,545). The N-terminal labeling of proteins using aderivative of the invention aids in the isolation, identification andselection of targets. This approach is also useful for the detection ofprotein interactions in ribosome/nucleic acid-protein display-basedprotein microarrays. Derivatives of the invention are thus particularlyuseful for the isolation, characterization and identification of proteintargets.

The use of in vitro lysate-based protein expression in phage display hasbeen described in published U.S. Application No. 2001/0029025. cDNA/mRNAlibraries are expressed in cell lysates with a derivative of theinvention and phage display libraries expressing cDNAs are screenedagainst the in vitro expressed proteins. Interacting proteins can beeasily identified by the N-terminal label without the need for cloningsteps. This approach is also useful in the selection of protein variantsin directed evolution. In addition, labeled protein synthesized in vitrousing a derivative of the invention can be employed to detect proteininteractions involving phage display, e.g., use with phage display-basedprotein microarrays/bead technologies. Multiplexing is also possible.

Another approach for directed evolution is described in published U.S.Application No. 2001/0039014, where in vitro transcription andtranslation in cell lysates is used for directed evolution of proteins.In this approach, the isolation, purification and characterization ofmutant proteins with improved functions could be readily accomplishedusing N-terminal labeling with a derivative of the invention.

Further, the labeled proteins may be introduced to cells, e.g., viaendocytosis, permeabilization or microinjection.

Mass spectrometry measures the mass of a molecule. The use of massspectrometry in biology is continuing to advance rapidly, findingapplications in diverse areas including the analysis of carbohydrates,proteins, nucleic acids and biomolecular complexes. For example, thedevelopment of matrix assisted laser desorption ionization (MALDI) massspectrometry (MS) has provided an important tool for the analysis ofbiomolecules, including proteins, oligonucleotides, andoligosacharrides. This technique's success derives from its ability todetermine the molecular weight of large biomolecules and non-covalentcomplexes (>500,000 Da) with high accuracy (0.01%) and sensitivity(subfemtomole quantities). Thus far, it has been found applicable indiverse areas of biology and medicine including the rapid sequencing ofDNA, screening for bioactive peptides and analysis of membrane proteins.

Surface plasmon resonance (SPR) may be used to study protein/proteininteractions. SPR is based on a change in the optical properties,particularly the refractive index of a surface after binding. Thischange, which can be measured very accurately, can then be used todetect both the extent and rate of binding. For example, incident lightstriking the back side of a thin gold layer, having a ligand monolayeron the front side, at variable angle penetrates into the ligandmonolayer. Interaction occurs with the surface plasmons and, at acertain angle, the reflected light is reduced to a minimum due toplasmon resonance. The position of the minimum is detected and permitscalculation of the refractive index. Binding of a molecule by the ligandchanges the refractive index. SPR has been used to measure adsorption ofproteins on polymer surfaces, protein binding to DNA, interactions ofproteins with self-assembled monolayers on gold surfaces, interactionsof proteins with phospholipid layers, and antibody-antigen interactions.For instance, a sensor chip with a carboxymethylated dextran matrix(Biocore) is pre-immobilized with streptavidin. Biotin labeled proteinsprepared by the methods of the invention are contacted with one or moreproteins and optical properties before and after contacting determined.

Electrophoresis may be employed to detect and/or isolate proteins or todetect the interaction of molecules with proteins which are translatedin a translation system. Many proteins are capable of simultaneouslyinteracting with multiple protein partners. For example, some proteinsmay have up to 86 proteins that they interact within the cell. The useof labeled proteins, e.g., labeled with affinity, fluorophore,luminescent or bioluminescent labels, in conjunction withelectrophoresis allows the observation of an unlimited number ofsimultaneous protein-protein and/or protein-nucleic acid interactions.

Thus, the methods of the invention allow for a large number of moleculesto be rapidly screened for possible interaction with the expressedprotein of specific genes, even when the protein has not been isolatedor its function identified. It also allows a library of proteinsexpressed by a pool of genes to be rapidly screened for interaction withmolecules, e.g., compounds, without the necessity of isolating theproteins. For example, a library of molecules can be screened toidentify those which serve as ligands for specific target protein. Themolecules might be part of a combinatorial library of compounds orpresent in a complex biological mixture such as natural samples whichmay contain therapeutic compounds. The molecules might interact with thenascent proteins by binding to them or to cause a change in thestructure or other property of the nascent protein by chemical orenzymatic modification.

Interaction of a specific molecule can be determined by comparing thepresence or absence of the nascent protein exposed to the specificmolecule with a similar analysis or measurement of the nascent proteinthat has not been exposed. The binding strength of the molecule can thenbe ascertained by altering the concentration of the specific moleculesadded to the protein synthesis system and measuring the change in therelative intensity of bands assigned to the uncomplexed and complexednascent protein. In addition to gel electrophoresis, which measures theelectrophoretic mobility of proteins in gels such as a polyacrylamidegel, the detection and/or isolation of complexed or noncomplexedproteins can be performed using capillary electrophoresis (CE) (see,e.g., U.S. Pat. No. 5,571,680 (Chen)). CE measures the electrophoreticmigration time of a protein which is proportional to the charge-to-massratio of the molecule.

In an embodiment, different labels are introduced to two or moreproteins of interest, e.g., introduced at the N-terminus of each proteinor at the N-terminus of one protein and internally on the other protein.For example, each distinct protein comprises a label that emits light ata wavelength that is different than the label on a different protein.The two labeled proteins are mixed under conditions favorable forbinding. The binding mixture is then subjected to CE and complexes ofthe two proteins, as well as the noncomplexed proteins, are detected. Inaddition, labeling of potential ligands of the protein of interest witha second label which is sensitive to the proximity of the first label,e.g., using FRET, BRET or LRET, permits the detection of the proximityof the two labels.

One form of CE, sometimes termed affinity capillary electrophoresis, hasbeen found to be highly sensitive to interaction of proteins with othermolecules including small ligands as long as the binding produces achange in the charge-to-mass ratio of the protein after the bindingevent. For example, the interaction of an antibody with the nascentproteins can be detected due to a change in the effectiveelectrophoretic mobility of the complex formed. However, the highestsensitivity may be obtained if the protein has a label with aspecifically detectable electromagnetic spectral property such as afluorescent dye. Detection of a peak in the electrophoresis chromatogramis accomplished by laser induced emission of mainly visible wavelengths.Examples of useful fluorescent dyes for CE include fluoroscein,rhodamine, Texas Red and BODIPY.

In addition to interactions that involve the binding of one or moremolecules to the labeled proteins, interactions that result in amodification of the labeled protein including but are not limited tophosphorylation, proteolysis, and glycosylation, can be detected usingelectrophoresis.

To determine the concentration of a protein of interest in a sample, thesample can be mixed with a corresponding labeled protein of interest anda protein that binds to both the labeled and unlabeled protein ofinterest. The mixture can then be subjected to CE and the concentrationof the protein of interest determined (see for example, U.S. Pat. No.5,571,680 (Chen)). This technique can also be used for the capture ofthe labeled and/or unlabeled protein, for example, using techniquessimilar to ELISA, for example, via capture by an antibody specific tothe cyanobenzothiazole, as in a “sandwich” ELISA. For examples of ELISA“sandwich” test procedures, see Schuurs and van Weemen, J. Immunoassay1980; 1:229-49.

General Synthetic Methods

Labels and detectable moieties, for example, those that are covalentlylinked to a cyanobenzothiazole or derivative thereof, permit the readydetection of that molecule in a complex mixture after reaction with apeptide of interest. The label may be one that is added to thecyanobenzothiazole core by chemical synthesis by the techniquesdescribed herein, or by those techniques well known to those of skill inthe art. For instance, the attachment of fluorescent or other labelsonto a core molecule can be accomplished by chemical modification. SeeGreg T. Hermanson, Bioconjugate Techniques, Academic Press, San Diego,Calif. (1996). Additional information regarding general syntheticmethods that may be used to prepare the compounds described herein maybe found in March's Advanced Organic Chemistry Reactions, Mechanisms,and Structure, 5^(th) Ed. by Michael B. Smith and Jerry March, JohnWiley & Sons, Publishers; and Wuts et al. (1999), Protective Groups inOrganic Synthesis, 3^(rd) Ed., John Wiley & Sons, Publishers.

The methods of preparing compounds of the invention can produce isomersin certain instances. Although the methods of the invention do notalways require separation of these isomers, such separation may beaccomplished, if desired, by methods known in the art. For example,preparative high performance liquid chromatography methods may be usedfor isomer purification, for example, by using a column with a chiralpacking.

The general methods for linking a cyanobenzothiazole to a linking groupY to form a compound of formula I are typically well known in the art.Such ‘linking’ or ‘coupling’ reactions are standard techniques.Techniques used to couple linking groups to various benzothiazolederivatives can be found in standard handbooks such as Hermanson'sBioconjugate Techniques. Of course, one skilled in the are wouldrecognize that compounds of formula I can be prepared by not only areaction between an appropriate cyanobenzothiazole and a group Y-X butalso by a reaction between a cyanobenzothiazole-Y group with anappropriately functionalized group X, such as a group X with anappropriate electrophile or nucleophile. For example, a primary hydroxylgroup on a linking group can be converted to a leaving group, such as atoluenesulfonyl group, which group can then be displaced with anucleophile, for example, a deprotonated 6′-hydroxycyanobenzothiazole.Specific examples of forming cyanobenzothiazole-Y groups are describedby Zhou (see J. Amer. Chem. Soc. 2006, 128(10), 3122).

Numerous succinimidyl esters that are useful for preparing compounds offormula I are commercially available, for example, from InvitrogenCorporation. Additionally, one skilled in the art can use commonlyreagents and conditions for preparing succinimidyl esters. Hermanson'sBioconjugate Techniques provides an extensive description of linkingreactions that can be used to prepare compounds of formula I,particularly in Part I, which describes “Functional Targets” and “TheChemistry of Reactive Groups” (pages 1-416). For example, commonreagents used to prepare succinimidyl esters includeN-hydroxysuccinimide (“NHS”, J. Am. Chem. Soc., 86:1839 (1964)) and acarbodiimide activating agent such as dicyclohexyl-carbodiimide (“DCC”)or 1,3-dimethylaminoprproply-ethylcarbodiimide (“EDC”; J. Am. Chem.Soc., 95:875 (1973)). Alternatively, a ‘self-activating’ NHS derivativecan be used, such as N-trifluoroacetyl-succinimide (“TFA-NHS”),N,N-disuccinimidyl carbonate (Tetrahedron Lett., 22:4817 (1981)), orO—(N-succinimidyl)-N,N,N′,N′-bis(tetramethylene)uraniumhexafluorophosphate. Depending on the reactivity and solubility of thebenzothiazole or linking group being activated, the conditions can rangefrom organic to aqueous solvents. For example, a suitable organicsolvent can be dimethylformamide (“DMF”). These reactions can be run inthe presence of a base, such as a hindered amine base, for example,triethylamine or diethylisopropylamine, whereas aqueous conditions mayinclude adjusting the pH to a range from about 6.5 to about 8.5.

When a group X-Y contains an amine, such as with a cyclic nucleotide, anucleic acid, or many chemotherapeutics and proteins, then asuccinimidyl ester of such a group can be used in the coupling reaction.When the group X contains an acid, such as with many quenchers,proteins, chemotherapeutics, and avidins, then the acid can be convertedto a succinimidyl ester and combined with an amine-terminated Y groupthat has been previously linked to a cyanobenzothiazole. Otheractivating groups, such as sulfosuccinimidyl esters, tetrafluorophenylesters, sulfodichlorophenol esters, isothiocyanates, sulfonyl chlorides,dichlorotriazines, aryl halides, or acyl azides can be used in place ofsuccinimidyl esters to link with amines. Furthermore, one skilled in theart can readily convert certain organic moieties to suitable amines oracids using standard transformations, including oxidations, reductions,and displacement reactions. Additionally, protecting groups can be usedto simplify the preparation of certain compounds of formula I. The useof protecting groups is well known in the art (see for example, see forexample, Greene, Protecting Groups In Organic Synthesis; Wiley: NewYork, 1981).

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the present invention could be practiced. It should be understoodthat many variations and modifications may be made while remainingwithin the scope of the invention.

EXAMPLES Example 1: Preparation of Cyanobenzothiazole Derivatives PartA. Synthesis of4-(3-(2-cyanobenzo[d]thiazol-6-yloxy)propylcarbamoyl)-2-(3-(dimethylamino)-6-(dimethyliminio)-6H-xanthen-9-yl)benzoate;“2-Cyano-(6-oxopropylamidotetramethyl-5′-carboxyrhodamine)benzothiazole”(compound 3028)

Method A:

A flask containing 100 mg of6-(N-Boc-3-aminopropyloxy)-2-cyano-benzothiazole (or “tert-butyl3-(2-cyanobenzo[d]thiazol-6-yloxy)propylcarbamate”, (W. Zhou, J. Amer.Chem. Soc. 2006, 128(10), 3122)) was stirred at 0° C. in dichloromethane(1 mL), trifluoroacetic acid (1 mL), and anisole (250 uL). After 2hours, solvent was evaporated. Ether (2 mL) was added to precipitate theproduct. The white solid was washed 2 times with 2 mL of diether etherand dried under vacuum. The solid was used without further purification.

To a flask containing the above solid was added 6-TAMRA SE (138 mg, 0.3mmol, 1 equiv) dissolved in 1 mL DMF and DIPEA (50 μl). After 24 hoursat room temperature, the solvent was removed in vacuo. The residue waseluted through silica in a 90% heptane/10% methanol eluent. Appropriatefractions were combine and evaporated. The film was dissolved in 1 mLacetone and precipitated with 6 mL diethyl ether to yield 10 mg solidcompound 3028. ¹H NMR (300 MHz, DMSO) δ 8.78 (t, 1H, J=5.6), 8.20 (td,2H, J=4.1, 8.3), 8.03 (d, 1H, J=9.0), 7.78 (s, 2H), 7.20 (dd, 1H, J=2.5,9.1), 6.92 (d, 5H, J=23.2), 4.09 (t, 2H, J=5.9), 3.41 (dd, 3H, J=6.1,11.9), 3.19 (s, 12H), 2.43 (d, 10H, J=1.7), 1.98 (dd, 2H, J=6.0, 12.1).

Alternatively, the compounds may be purified by preparative reversephase HPLC.

Analogous compounds with fluorescein, Alexa 633, biotin, and IC-5 labelswere synthesized using method A by substituting the TAMRA-SE for theappropriate FAM-SE (Sigma), biotin-SE (Sigma), Alexa-633-SE(Invitrogen), or IC-5-SE (Biosearch Technologies, Cat. No. FC-1065S-25).As would be readily recognized by one skilled in the art, similartechniques can be used to prepare cyanobenzothiazole derivatives linkedto other groups of interest, including reporter moieties, affinitylabels, quencher moieties, photocrosslinking moieties, or solidsupports.

Part B

The following compounds were synthesized using Method A utilizing theappropriate 5,6FAM-SE, Bodipy488-SE, biotin-SE, or IC-5-SE in place of6-TAMARA SE.

4(and5)-(3-(2-cyanobenzo[d]thiazol-6-yloxy)propylcarbamoyl)-2-(3-hydroxy-6-oxo-6H-xanthen-9yl)benzoicacid (e.g., compound 3066)

Mixture of isomers (66%:35%); ¹H NMR (300 MHz, DMSO) δ 10.12 (s), 8.90(t), 8.76 (t), 8.44 (d), 8.22 (dd), 8.11 (m), 7.89 (d), 7.82 (d), 7.66(s), 7.33 (m), 7.21 (dd), 6.66 (d), 6.54 (m), 4.18 (t), 4.08 (t), 3.50(dd), 3.37 (t), 2.07 (m), 1.97 (m), 1.22 (s), 0.83 (t). MS: Calcd forC₃₂H₂₁N₃O₇S, 592.1; found 592.

(Z)—N-(3-(2-cyanobenzo[d]thiazol-6-yloxy)propyl)-3-(1-(difluoroboryl)-5-((3,5-dimethyl-2H-pyrrol-2-ylidene)methyl)-1H-pyrrol-2-yl)propanamide(compound 3226)

¹H NMR (300 MHz, DMSO) δ 8.17 (d, 1H, J=9.1), 8.07 (t, 1H, J=5.7), 7.87(d, 1H, J=2.5), 7.67 (s, 1H), 7.35 (dd, 1H, J=2.5, 9.1), 7.08 (d, 1H,J=3.9), 6.56 (s, OH), 6.39 (d, 1H, J=4.0), 6.33 (s, 1H), 4.12 (t, 2H,J=6.3), 3.30 (dd, 2H, J=6.4, 12.2), 3.12 (t, 2H, J=7.5), 2.50 (s, 3H),2.28 (s, 3H), 1.95 (p, 2H, J=6.4). MS Calcd for C₂₅H₂₄BF₂N₅O₂S, 508;found 507.

N-(3-(2-cyanobenzo[d]thiazol-6-yloxy)propyl)-5-((3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide(compound 3167)

¹H NMR (300 MHz, DMSO) δ 8.18 (d, 1H, J=9.1), 7.92 (dd, 2H, J=4.1, 6.2),7.36 (dd, 1H, J=2.6, 9.1), 6.44 (s, 2H), 4.32 (dd, 1H, J=4.4, 7.7), 4.14(m, 3H), 3.26 (q, 2H, J=6.5), 3.10 (m, 1H), 2.83 (dd, 1H, J=5.1, 12.4),2.60 (d, 1H, J=12.3), 2.10 (t, 2H, J=7.3), 1.94 (t, 2H, J=6.4), 1.52 (m,4H), 1.33 (m, 2H). MS Calcd for C₂₁H₂₅N₅O₃S₂ 460.1; found 460.4.

2-((1E,3E,5E)-5-(1-(6-(3-(2-cyanobenzo[d]thiazol-6-yloxy)propylamino)-6-oxohexyl)-3,3-dimethylindolin-2-ylidene)penta-1,3-dienyl)-1-ethyl-3,3-dimethyl-3H-indoliumchloride (compound 3272)

¹H NMR (300 MHz, DMSO) δ 8.31 (t, 2H, J=13.1), 8.11 (d, 1H, J=9.1), 7.84(m, 2H), 7.60 (d, 2H, J=7.0), 7.29 (m, 6H), 6.55 (t, 1H, J=12.3), 6.26(dd, 2H, J=4.0, 13.8), 4.08 (m, 7H), 3.84 (s, 15H), 3.54 (s, OH), 3.19(d, 2H, J=5.9), 2.49 (dt, 4H, J=1.8, 3.7), 2.30 (s, OH), 2.05 (m, 2H),1.85 (m, 2H), 1.66 (d, 12H, J=2.8), 1.52 (dd, 2H, J=7.3, 14.7), 1.33(dd, 2H, J=7.3, 14.9), 1.24 (t, 3H, J=7.1). MS Calcd forC₄₄H₅₀N₅O₂S+712.4; found 712.

Part C. Synthesis of4-(6-(2-cyano-5-fluorobenzo[d]thiazol-6-yloxy)hexylcarbamoyl)-2-(3-(dimethylamino)-6-(dimethyliminio)-6H-xanthen-9-yl)benzoate(compound 3086)

5-Fluoro-6-hydroxybenzo[d]thiazole-2-carbonitrile (200 mg) was heated to65° C., 50 W, 40 minutes in a microwave with acetone (2 mL), potassiumcarbonate (284 mg), and tert-butyl 6-bromohexylcarbamate (265 μL).Afterward, an additional 150 μL tert-butyl 6-bromohexylcarbamate wasadded and reaction was heated to 80° C., 75 W, for 23 minutes. Thereaction was partitioned between ethyl acetate and bicarbonate, washedwith aqueous citric acid and brine, and evaporated. The crude materialeluted through a silica column with a mixture of heptane:ethyl acetate(3:1). Yield 78%.

tert-Butyl 6-(2-cyano-5-fluorobenzo[d]thiazol-6-yloxy)hexylcarbamate(200 mg) was added to cold (0° C.) solution of dichloromethane (3 mL),trifluoroacetic acid (3 mL), and anisole (300 μL). After 15 minutes, themajority of solvent was evaporated, and 30 mL of diethyl ether wasadded. The precipitate was isolated (165 mg).

6-(6-Aminohexyloxy)-5-fluorobenzo[d]thiazole-2-carbonitrile (50 mg) wasstirred with 6-TAMRA-SE (65 mg) as in method A above. Yield 10 mg. ¹HNMR (300 MHz, DMSO) δ 8.67 (t, 1H, J=5.8), 8.17 (q, 2H, J=8.2), 8.05(dd, 2H, J=9.7, 21.1), 7.78 (s, 1H), 6.90 (d, 5H, J=26.3), 4.08 (t, 2H,J=6.4), 3.17 (s, 11H), 1.73 (m, 2H), 1.48 (m, 2H), 1.35 (s, 4H). MSCalcd for C₃₉H₃₆FN₅O₅S, 706.2; found 706.

Part D. Synthesis of4-(6-(2-cyano-7-nitrobenzo[d]thiazol-6-yloxy)hexylcarbamoyl)-2-(3-(dimethylamino)-6-(dimethyliminio)-6H-xanthen-9-yl)benzoate(compound 3087)

6-Hydroxybenzo[d]thiazole-2-carbonitrile (352 mg) was heated in amicrowave with ZrO(NO₃)₂×H₂O (462 mg) and acetone (7 mL) at 100° C. (200W) for 10 min. Product was extracted with dichloromethane and elutedthrough silica with heptane:ethyl acetate (1:1). Yield 222 mg.

6-Hydroxy-7-nitrobenzo[d]thiazole-2-carbonitrile (100 mg) was heated to70° C. at 50 W for 30 minutes in a microwave with acetone (2 mL),potassium carbonate (125 mg), and tert-butyl 6-bromohexylcarbamate (139mg). After which an additional 150 μL tert-butyl 6-bromohexylcarbamatewas added and reaction was heated to 80° C., 75 W, for 30 minutes. Afterwhich an additional 300 μL tert-butyl 6-bromohexylcarbamate, cesiumcarbonate (162 mg) and diglyme (1 mL) was added and reaction was heatedto 100° C., 75 W, for 250 minutes. The reaction was partitioned betweenethyl acetate and bicarbonate, washed with aqueous citric acid andbrine, and evaporated. The crude material eluted through a silica columnwith a mixture of heptane:ethyl acetate (2:1). Yield 44%

tert-Butyl 6-(2-cyano-7-nitrobenzo[d]thiazol-6-yloxy)hexylcarbamate (50mg) was added to cold (0° C.) solution of dichloromethane (1 mL),trifluoroacetic acid (1 mL), and anisole (99 μl). After 30 minutes, themajority of solvent was evaporated, and 30 mL of diethyl ether wasadded. The precipitate was isolated and used without furtherpurification.

6-(6-Aminohexyloxy)-7-nitrobenzo[d]thiazole-2-carbonitrile (51 mg) wasstirred with 6-TAMRA-SE (50 mg) as in Method A above. Yield 13 mg. ¹HNMR (300 MHz, DMSO) δ 8.72 (t, 1H), 8.58 (d, 1H, J=7.3), 8.22 (d, 2H,J=8.0), 7.83 (s, 2H), 6.98 (d, 5H), 4.39 (t, 2H), 3.24 (s, 15H), 1.82(m, 2H), 1.52 (m, 4H), 1.40 (m, 2H). MS Calcd for C39H36N6O7S, 733.2;found 733.6.

Part E. Synthesis of4-(6-(2-cyanobenzo[d]thiazol-6-ylamino)-6-oxohexylcarbamoyl)-2-(3-(dimethylamino)-6-(dimethyliminio)-6H-xanthen-9-yl)benzoate(compound 3082

6-(tert-Butoxycarbonylamino)hexanoic acid (316 mg) was mixed withanhydrous THF (10 mL), 6-aminobenzo[d]thiazole-2-carbonitrile (200 mg),iso-butylchloroformate (193 μl), and N-methylmorpholine (314 μl) at −4°C. The reaction was allowed to stir overnight at RT. The reaction waspartitioned between ethyl acetate and bicarbonate. The ethyl acetatelayer was evaporated, and the residue was eluted through silica withheptane:ethyl acetate (1:2). Yield 354 mg.

tert-Butyl 6-(2-cyanobenzo[d]thiazol-6-ylamino)-6-oxohexylcarbamate (350mg) was added to cold (0° C.) solution of dichloromethane (4 mL),trifluoroacetic acid (4 mL), and anisole (400 μl). After 135 minutes,the majority of solvent was evaporated, and 10 mL acetonitrile and 30 mLof diethyl ether was added. The mixture was allowed to sit overnight.The precipitate was isolated and used without further purification.

6-Amino-N-(2-cyanobenzo[d]thiazol-6-yl)hexanamide (100 mg) was stirredwith 6-TAMRA-SE (122 mg) as in method A above. Yield (22 mg). ¹H NMR(300 MHz, DMSO) δ 10.31 (s, 1H), 8.67 (dd, 2H, J=3.8, 7.2), 8.12 (m,3H), 7.77 (s, 1H), 7.63 (dd, 1H, J=2.1, 9.1), 3.17 (s, 14H), 2.31 (t,2H, J=7.4), 1.57 (m, 2H), 1.47 (m, 2H), 1.31 (m, 2H). MS: Calcd forC₃₉H₃₆N₆O₅S: 701.2; found 701.6.

Example 2: Synthesis of(E)-N-(2-(2-amino-3-mercaptopropanamido)ethyl)-4-((4-(dimethylamino)phenyl)diazenylbenzamide(compound 3191)

(E)-2,5-Dioxopyrrolidin-1-yl4-((4-(dimethylamino)phenyl)diazenyl)-benzoate (200 mg) was mixed withdimethyl formamide (5 mL), tert-butyl1-(2-aminoethylamino)-1-oxo-3-(tritylthio)propan-2-ylcarbamate (333 mg),and diisopropylethylamine (285 μl). After 12 hours, the reaction waspartitioned between ethyl acetate and aqueous citric acid. The organiclayer was washed with bicarbonate and, then, brine. After evaporation,the residue was eluted through silica with heptane:ethyl acetate (1:1).Yield: 242 mg.

(E)-tert-Butyl1-(2-(4-((4-(dimethylamino)phenyl)diazenyl)benzamido)-ethylamino)-1-oxo-3-(tritylthio)propan-2-ylcarbamate(240 mg) was added to a cold solution of trifluoroacetic acid (10 mL),water (500 μl), and triisopropylsilane (100 μl). After 5 hours, diethylether (50 mL) was added to precipitate product, compound 3191. Theproduct was further purified by preparative reverse phase HPLC. Yield 60mg. ¹H NMR (300 MHz, DMSO) δ 8.56 (m, 2H), 8.18 (s, 3H), 7.92 (d, 2H,J=8.5), 7.75 (d, 4H, J=8.3), 6.78 (d, 2H, J=8.2), 3.85 (m, 1H), 3.35 (m,3H), 3.20 (m, 1H), 3.01 (s, 6H), 2.85 (m, 2H), 2.50 (s, 1H).

Example 3: N-Terminal Peptide Labeling with a CyanobenzothiazoleDerivative

In this example, a cyanobenzothiazole-rhodamine reagent is added tosolutions containing various concentrations of materials containingcysteine residues. The solutions are incubated with thecyanobenzothiazole-rhodamine reagent under conditions where an adductcan be formed between the N-terminal cysteine residue and the reagent.After incubation with the reagent, the presence of a new, labeledspecies is detected by fractioning a small portion of the reactionmixture onto a silica thin layer chromatography (TLC) plate andexamining the presence of a fluorescent species.

Five different materials containing cysteine residues were reacted witha cyanobenzothiazole-rhodamine reagent. Tocinoic acid (Sigma) is apeptide containing an amino and carboxy terminal cysteine residueconnected by a disulfide bond (peptide sequence:Cys-Tyr-Ile-Gln-Asn-Cys) (SEQ ID NO:7). To reduce the disulfide bonds inthe peptide, 50 μl of 1 mM tocinoic acid was mixed with 5 μl 1M HEPES pH8.0, 0.25 μl Bond Breaker (Pierce Chemical Co) and 46 μl water. Tocreate an oxidized Tocinoic acid solution, 50 μL of tocinoic acid (1 mM)was mixed with 5 μL 1M HEPES pH 8.0 and 45 μl water. Bachem H4696(Bachem Bioscience Inc., King of Prussia, Pa.) is a peptide(Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys) (SEQ ID NO:8) containing an internalcysteine residue. A working solution of Bachem H4696 was made by mixing48 μl 5 mM Bachem H4696 with 0.25 μl Bond Breaker and 1 μl HEPES pH 8.0.Bachem H4702 is a peptide (Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr) (SEQ IDNO:9) containing an N-terminal cysteine residue. A working solution ofBachem H4702 was made by mixing 48 μl 5 mM Bachem H4702 with 0.25 μlBond Breaker and 1 μl 1M HEPES pH 8.0. Working solutions of 10 mMCys-Gly dipeptide (Sigma; 15 mg) and 20 mM Cysteine (Sigma) were alsomade.

A reaction buffer for the cyanobenzothiazole labeling reaction was madeby mixing 500 μL 1M HEPES pH 7.5 with 7.5 mL water. Six sets ofreactions were set up to allow various molar ratios of peptide toreagent to be tested. Each set contained six reactions which would eachcontain a different cysteine material. The relative molar ratios ofpeptide to reagent tested varied from 0.3:1 to 2:1. To all reactions, 75μl of reaction buffer was added. To each reaction in each set, adifferent amount of water was added: 16.7 μl to the first set, 13.3 μlto the second set, 10 μl to the third set, 6.7 μl to the fourth set and3.3 μl to the fifth set. The sixth set of reaction tubes would not haveany water added as it would constitute the highest ratio of cysteinematerial to cyanobenzothiazole reagent. One reaction tube was set up asa no peptide control in which 20 μl water was added.

Diluted solutions of cysteine (2.5 μl 20 mM cysteine to 100 μl withwater), Bachem H4696 (10 μl of the above solution to 100 μl with water),Bachem H4702 (10 μl of the above solution to 100 μl with water) andCys-Gly (5 μl of above solution to 100 μl with water) were added todifferent reaction tubes in each of the six sets to final reactionvolumes of 95 μl. The oxidized tocinoic acid and reduced tocinoic acidwere also added to different reaction tubes in each of the six sets tofinal reaction volumes of 95 μl. In total, each reaction tube in each ofthe 6 sets contained a different cysteine solution.

To all reaction tubes, 5 μl of 1 mM cyanobenzothiazole-rhodamine reagentwas added and mixed. After a 15 minute incubation at room temperature, 1μl from each reaction tube was spotted onto a silica gel TLC plate. Theplate was developed in a mixture of 90 parts EtOH, 10 parts water andone part glacial acetic acid. After development, the plate was airdried, visualized under UV light to confirm the reaction proceeded tocompletion.

The no peptide control reaction containing only buffer and thecyanobenzothiazole-rhodamine reagent gave a strong fluorescent spot atan Rf value of ˜0.8 and a weak spot at an Rf value of ˜0.4. Thisidentifies the mobility of the original, unreacted cyanobenzothiazolereagent and demonstrates the reagent is unaffected by the reactionconditions.

FIG. 3 shows an image of a thin layer chromatography (TLC) platecaptured on an Ambis Imaging system set to detect the fluorescentemission from fluorescent species present on the TLC plate when exposedto ultraviolet light and collected through a filter excludingultraviolet light present on the imaging camera.

Reactions wherein increasing amounts of cysteine was used (lanes 2-7)show the presence of a new, strong fluorescence adduct with a mobilityat an Rf value of ˜0.42 along with the presence of low amounts of otherspecies (adduct). The amount of new fluorescent species increase untilthe molar amount of the cysteine and benzothiazole are approximatelyequal (lanes 4-5). This demonstrates that 1) the reaction betweencysteine and the cyanobenzothiazole-rhodamine reagent is rapid (˜15minutes), and 2) the reaction requires about 1 mole equivalent cysteineto react with 1 mole of cyanobenzo-thiazole reagent.

Reactions wherein increasing amounts of Bachem H4696 (peptide with aninternal cysteine) was used (lanes 8-13) showed a fluorescent mobilitypattern essentially the same as was seen with the cyanobenzothiazolereagent alone (lane 1). This most likely demonstrates 1) either internalcysteines are unable to react with the cyanobenzothiazole reagent, 2) alarger amount of the peptide is needed to react with thecyanobenzothiazole reagent, or 3) any adduct which does form is unstableand reverts back to the starting material readily making detection ofthe adduct almost impossible.

In contrast, reactions where increasing amounts of Bachem H4702 (peptidecontaining an N-terminal cysteine) showed a significant amount of adductwith very low mobility (lanes 14-19). The amount of new adduct formationincreased with a corresponding loss of unreacted cyanobenzothiazolereagent. Therefore, the cyanobenzothiazole reagent is able to form astable adduct with a peptide with an N-terminal cysteine. This is mostlikely caused by the formation of a cyclic benzothiazole product throughthe attack of the N-terminal amino group on the amino acid analogous tothat seen with the formation of luciferin through the reaction withcysteine. However, if the N-terminal cysteine group is involved in adisulfide bond such as in the reaction with the oxidized tocinoic acid(lanes 20-25), no new adduct is formed and no loss of unreactedcyanobenzothiazole reagent is seen with increasing amounts of peptide.

If the disulfide bond in tocionic acid is reduced to free cysteine(reduced tocinoic acid above), the resulting N-terminal cysteine isavailable to react with the cyanobenzothiazole reagent (lanes 26-31) anda new adduct with low mobility is formed. The new adduct is formed in amanner similar to that seen in the cysteine reaction i.e. the amount ofadduct formed is dependent on a ratio of 1 mole equivalent reducedtocinoic acid to 1 mole cyanobenzothiazole reagent. Reactions with thedipeptide Cys-Gly (lanes 32-37) also demonstrate the rapid labeling of aN-terminal cysteine by the cyanobenzothiazole reagent even in a smallpeptide.

Therefore, this example demonstrates that the cyanobenzothiazole reagentcan readily label a peptide, large or small, containing an availableN-terminal cysteine.

Example 4: Labeling Proteins with Cyanobenzothiazole Reagents

In this example, a protein with an N-terminal cysteine residue waslabeled with a compound of the invention. The amount of labeling wascompared to other proteins in the solution, and to a parallel reactionwhere the target protein was replaced with a protein identical to thetarget protein except that the N-terminal cysteine residue was exchangedfor an alanine residue. The example demonstrates that: 1) a fusionprotein construct can be constructed such that, when digested with TEVprotease, it generates a protein of interest with an N-terminalcysteine; 2) exposure of a protein with an N-terminal cysteine residue,such as that produced by digestion of a properly designed fusion proteinconstruct with TEV protease, will become highly fluorescent when exposedto a compound of the invention containing a fluorescent moiety, eventhough other proteins in the reaction without N-terminal cysteineresidues gain little or no fluorescence, and; 3) a parallel reactionthat contains a TEV digested fusion construct identical to the one thatexposes a cysteine residue upon digestion, but which exposes an alanineresidue, will show very little or no labeling of the proteins in thesolution, including the protein identical to the protein having anN-terminal cysteine but where the N-terminal cysteine has been replacedwith an alanine.

To demonstrate these points, an experiment—which is described in detailbelow—was performed having the following steps: A) recombinant DNAclones were constructed designed to express a fusion protein in E. colihaving: i) an affinity protein tag [GST] at the N-terminus of the intactfusion construct for rapid and easy purification of the fusion proteinspecies followed by, ii) a protein sequence encoding a TEV proteaserecognition site followed by, iii) another protein segment. Twoconstructs were created that differed only in that one construct wouldexpose a cysteine residue at the new N-terminus of the protein producedby TEV cleavage of the fusion construct, whereas the other constructwould expose an alanine residue at the new N-terminus; B) expression ofthe recombinant DNAs in E. coli and confirmation that the bacteriaexpressed the fusion proteins; C) purification of the fusion proteinsfrom an E. coli lysate by employing the affinity protein tag, and; D)digestion of the fusion constructs followed by exposure of both intactand cleaved fusion protein to PBI compound 3128 (see FIG. 2A), followedby analysis to detect the labeling of protein species in the reactionmixtures.

Step A). Construction of Recombinant DNA Species Encoding the FusionProtein Pairs.

Plasmid species were designed that encoded a prokaryotic promoter andtranslation initiation region followed in frame with the coding sequenceof glutathione S transferase [GST, an affinity protein tag]. One versionhad the GST followed in frame by a coding sequence that contained arecognition sequence for TEV protease followed by a cysteine followed inframe with other proteins. A second plasmid species was designed to beidentical to the plasmid above but with the encoded cysteine followingthe TEV site replaced with an alanine. The protein coding regions ofbeetle luciferase was then fused in frame with the end of the codingsequence encoding the TEV protease site such that there was onecontinuous coding region that encoded all of these polypeptide segments.

The plasmids were confirmed by DNA sequence analysis.

Amino Acid and Nucleotide Sequences for GST-Luc (Ala and Cys):

Amino Acid Sequence GST-(TEV-Cys)-Luc

(SEQ ID NO: 10) MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSGGGGGENLYFQCIAMEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKI REILIKAKKGGKSKLVNucleotide Sequence for GST-(TEV-Cys)-Luc

(SEQ ID NO: 11) atgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgagggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttgaatggtgatcatgtaacccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgtttcaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatccggaggtggtggcggagaaaacctgtacttccaatgcatcgccATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCTCTAGAGGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAATTCCTCTGGATCTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTACGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTTCATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAAACGCTTCCATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTATGTAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTTGAAGTCTTTAATTAAATACAAAGGATATCAGGTGGCCCCCGCTGAATTGGAATCGATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCCCGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACGATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAAAAAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGAAAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGTCCAAATTGgttt AAAmino Acid Sequence for GST-(TEV-Ala)-Luc

(SEQ ID NO: 12) SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYVIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSGGGGGENLYFQAIAMEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIR EILIKAKKGGKSKLVNucleotide Sequence for GST-(TEV-Ala)-Luc

(SEQ ID NO: 13) atgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttgaatggtgatcatgtaacccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgtttcaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatccggaggtggtggcggagaaaacctgtacttccaagcgatcgccATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCTCTAGAGGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAATTCCTCTGGATCTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTACGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTTCATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAAACGCTTCCATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTATGTAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTTGAAGTCTTTAATTAAATACAAAGGATATCAGGTGGCCCCCGCTGAATTGGAATCGATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCCCGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACGATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAAAAAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGAAAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGTCCAAATTGgtt tAStep B). Expression of Fusion Proteins.

Cultures of bacteria transformed with the confirmed plasmids were grownand induced for protein expression. After growth of the cultures,expression of the generation of a fusion protein of the expected sizewas confirmed by SDS PAGE fractionation of a sample of the cells withCoomassie Blue staining to detect the protein bands. Expression of thefusion proteins was estimated to be 1-5% of the total soluble protein inthe cell lysates.

Step C). Purification of the Fusion Proteins.

After growth of the cultures, the cells were collected by centrifugationand frozen at −20° C. until ready for processing. Once ready forpurification, the cell pellets were thawed and resuspended in a buffer A(1×PBS, pH 7.3, 1 mM PMSF, 1 Roche complete protease tablet per 50 mL),and cells were resuspended in this buffer at a ratio of 8-10 mL ofbuffer per gram cell paste. The cells were then lysed by sonication andinsoluble cell debris precipitated by centrifugation of the lysed cellsat 3900×G for 10 minutes at 4° C.

After centrifugation, the supernatant above the pellet was carefullyremoved and applied to a column of Glutathione sepharose (from GEHealthcare) equilibrated in 1×PBS, pH 7.3. After application, the columnwas washed with 10-20 column volumes of 1×PBS buffer (pH 7.3), then asolution containing 10-15 mM glutathione in 50 mM Tris-HCl buffer (pH8.0) was applied to elute the protein. Fractions of the eluted materialswere collected during this process and a small amount of the fractionswere analyzed by SDS PAGE. As expected, the fusion proteins were greatlyenriched in the fractions where the column buffer containing glutathionewere eluting from the column. The fractions with the greatly enrichedfusion protein were pooled and dialyzed against 10 mM HEPES buffer (pH7.5, 50 mM NaCl).

Step D). Digestion and Labeling of the Fusion Proteins.

The protein concentration of the dialyzed fusion proteins weredetermined by use of Pierce's Coomassie Plus protein reagent per themanufacturer's protocol. Equal amounts of the paired protein constructswere diluted to ˜1 uM in ProTEV Protease buffer. ProTEV was added andfusion proteins were digested overnight at 4° C. ProTEV buffer was 50 mMHEPES, pH 7.0, 0.5 mM EDTA, 1 mM DTT. The samples of the digests wereanalyzed by SDS PAGE. The fusion construct with an alanine following theTEV cleavage site and the construct with a cysteine following the sitewere both digested to greater than 90% based upon the appearance of newprotein species the expected size for cleavage of the constructs at theTEV site and disappearance of the intact fusion protein band.

Samples of the two digests were then placed in fresh tubes containing 10mM HEPES, pH 7.5, and a sample of PBI compound 3028 added from a 2 mMacetonitrile stock (as in FIG. 3; DMSO can also be used) to produce afinal concentration of 10 μM PBI 3028. When using DMSO as a solvent, a6.5 mM stock solution was used. At set time intervals, samples of theselabeling reactions were added to new tubes containing a reagent toterminate the labeling reaction by reacting with the cyanobenzothiazolemoiety of the labeling reagent; this solution contained cysteine-HCl(final concentration 1-5 mM in stop reactions) and an equalconcentration of TCEP. It has also been found that a 10-fold lowerconcentration of TCEP is even more effective. This stop solution shouldbe made in ˜200 mM HEPES pH 8.0 in order to reduce acidity and preventprecipitation of proteins. Previous research had shown that reaction ofthis solution with the reagent rapidly converted the labeling reagent tothe desired chemical species.

After all the timed samples were collected, they were analyzed by SDSPAGE electrophoresis followed by imaging on the Typhoon. After imaging,the gels were stained with SimplyBlue SafeStain™ (Invitrogen) tovisualize the protein bands. A comparison of the gel images obtainedfrom fluorescent scanning of the gel prior to Coomassie staining andpost-Coomassie staining was performed (Coomassie stained gel not shown).The fluorescent gel image is shown in FIG. 4. As seen in the fluorescentgel image, the Cys N-terminal protein partner in the incubations of thebenzothiazole dye conjugate became highly fluorescent whereas the otherprotein species in these reactions, which are visible on the Coomassiestained gel, underwent little or no labeling at all (digested Cys afterTEV samples vs. digested Ala after TEV). In addition, the reactionswhere cleavage with TEV protease resulted in the new protein specieshaving an amino terminal alanine did not become highly fluorescentlylabeled. Finally, very little or no labeling is seen where the fusionprotein constructs are not digested with TEV protease (uncut Ala versionand Cys versions of the fusion protein). Thus, the cysteine residue thatallows strong labeling of the digested fusion protein construct does notbecome highly labeled if it is exposed to the reagent at an internalcysteine residue. The large dark spot on the bottom right of the figurearises from the colored protein standard loaded in that lane.

These observations demonstrate that there is at least a very strongpreference for labeling of an N-terminal cysteine residue with thecompounds of the invention, if not completely specific labeling. Also,the labeling is dependent upon the N-terminal residue of the proteinbeing a cysteine residue and such a protein can be generated bydigestion of a fusion protein construct by a protease.

Example 5: Confirmation of N-Terminal Labeling of Proteins FluorescentlyTagged with a Cyanobenzothiazole Labeling Reagent

In this example, a very specific fusion protein construct is labeledwith various cyanobenzothiazole labeling reagents. The protein is thenexposed to a second site-specific protease that cleaves the TEV digestedprotein a second time only a few amino acids from the new aminoterminus. This cleavage allows for the examination of the specificity oflabeling achieved prior to the second digestion. Accordingly, all of thefluorescence on digested protein should be eliminated by action of thesecond protease if the protein is exclusively labeled at the new aminoterminus. If the protein is labeled at multiple sites, however,treatment of the labeled protein with the second protease will generatea second protein species slightly smaller than the initial product thatis still highly fluorescent.

A fusion construct was produced that had a double protease cleavage sitebetween two protein partners with the order of segments being GST-TEVprotease site-Cys-Factor Xa protease site-HaloTag (version 2). Thisprotein was expressed in E. coli and purified by use of an affinityresin for GST fusion proteins (typically GE Healthcare Glutathionesepharose 4 fast flow) according to the instructions of the supplier.

The purity of the isolated fusion protein was examined by SDS PAGEelectrophoresis and found to contain a large amount of the desired fulllength protein. This protein was dialyzed and then digested with ProTEVprotease. Following cleavage, samples of the digest were labeled withcyanobenzothiazole labeling agents with different attached dye segments.A separate sample was labeled with the HaloTag TMR ligand, which hasbeen shown to label HaloTag protein well within the protein sequence.For information on HaloTag® technology and use of the HaloTag TMRligand, see the Technical Manual HaloTag® Technology: Focus on Imaging,Part # TM260, available from Promega Corporation; and M. Urh et al.,“Halolink™ Resin For Protein Pull-Down And Analysis” Cell Notes 2006,14, 15-19, available from Promega Corporation.

After labeling, samples of the purified protein labeled with each agentwere digested with Factor Xa. Following Factor Xa digestion, samples ofthe undigested labeled protein and Factor Xa digested labeled proteinwere fractionated on an SDS PAGE gel and the gel was imaged on a Typhoonimager. After imaging, the gel was stained with Coomassie brilliant blueto visualize the proteins using a method that would not rely on thefluorescence of the protein. The images of the fluorescent gel imagesand Coomassie stained gels are shown in FIG. 5A, FIG. 5B, and FIG. 5C.

As expected, when the fusion construct was labeled with HaloTag ligand(thus labeling the protein partner expected to be labeled with thecyanobenzathiazole reagents, but attaching the label at approximately inthe middle of the protein segment) and digested with Factor Xa, thelabeled protein only changed slightly in size and retained itsfluorescence (first two lanes of gel in FIG. 5A, FIG. 5B, and FIG. 5C).However, when the samples labeled with the cyanobenzathiazole reagentswere digested, almost all of the fluorescence associated with thelabeled protein band was eliminated from the protein, except for a verysmall amount of fluorescence associated with a protein species with themobility of the initial labeled protein (pairs of lanes illustratingloss of fluorescence in FIG. 5A, FIG. 5B, and FIG. 5C denoted 3028 TMR,3168 Alexa, and 3272).

When this segment of the gel was stained with Comassie, a large amountof protein slightly smaller than the labeled protein was found to bepresent in the lanes where the fusion protein was treated with Factor Xa(bottom panel, FIG. 5A, FIG. 5B, and FIG. 5C). Because this protein bandwas not fluorescent, yet arose from removal of only a few amino acidsfrom the amino terminus of the labeled protein, the fluorescent labelmust have been on the few amino acids digested off the construct—theamino terminus of the labeled protein species.

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 6, and FIG. 7 illustrate cleavageresults and representative gels of the results expected from labelingexclusively at the N-terminus, and non-specific labeling at cysteineresidues, respectively.

If a cyanobenzothiazole labeling reagent labels only the N-terminus andnot internal cysteines as illustrated in FIG. 6, one would expect to seethe cyanobenzothiazole label on the protein of interest to be removedwith cleavage of the protein at a protease site downstream from thelabel. An internal label, such as a HaloTag ligand control, would not beremoved with cleavage of the protein with the second protease. Afluorescent scan of an SDS-PAGE gel of these samples would show thedisappearance of fluorescence from the cyanobenzothiazole labeled bandafter cleavage with the second protease. The HaloTag ligand labeledprotein would show a shift in size but would remain fluorescent becausethe fluorescent label was not removed, which is illustrated in thefluorescent gel diagram of FIG. 6.

If the cyanobenzothiazole labeling reagent attaches to internalcysteines as illustrated in FIG. 7, one would expect the cleavage of thelabeled protein with a second protease to leave a fluorescent band of alower molecular weight, which would appear substantially similar to aHaloTag ligand labeled control protein, which is illustrated in thefluorescent gel diagram of FIG. 7.

Amino Acid Sequence for GST-(TEV-Cys-FXa)-HaloTag

(SEQ ID NO: 14) MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSGGGGGENLYFQCIAMIEGRAMGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYLWRNIIPHVAPSHRCIAPDLIGMGKSDKPDLDYFFDDHVRYLDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIACMEFIRPIPTWDEWPEFARETFQAFRTADVGRELIIDQNAFIEGALPMGVVRPLTEVEMDHYREPFLKPVDREPLWRFPNELPIAGEPANIVALVEAYMNWLHQSPVPKLLFWGTPGVLIPPAEAARLAESLPNCKTVDIGPGLFLLQEDNPDLIGSEIAR WLPGLVNucleotide Sequence for GST-(TEV-Cys-FXa)-HaloTag

(SEQ ID NO: 15) atgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttgaatggtgatcatgtaacccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgtttcaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatccggaggtggtggcggagaaaacctgtacttccaatgcatcgctatgatagagggtagagctatgggatccgaaatcggtacaggcttccccttcgacccccattatgtggaagtcctgggcgagcgtatgcactacgtcgatgttggaccgcgggatggcacgcctgtgctgttcctgcacggtaacccgacctcgtcctacctgtggcgcaacatcatcccgcatgtagcaccgagtcatcggtgcattgctccagacctgatcgggatgggaaaatcggacaaaccagacctcgattatttcttcgacgaccacgtccgctacctcgatgccttcatcgaagccttgggtttggaagaggtcgtcctggtcatccacgactggggctcagctctcggattccactgggccaagcgcaatccggaacgggtcaaaggtattgcatgtatggaattcatccggcctatcccgacgtgggacgaatggccagaattcgcccgtgagaccttccaggccttccggaccgccgacgtcggccgagagttgatcatcgatcagaacgctttcatcgagggtgcgctcccgatgggggtcgtccgtccgcttacggaggtcgagatggaccactatcgcgagcccttcctcaagcctgttgaccgagagccactgtggcgattccccaacgagctgcccatcgccggtgagcccgcgaacatcgtcgcgctcgtcgaggcatacatgaactggctgcaccagtcacctgtcccgaagttgttgttctggggcacacccggcgtactgatccccccggccgaagccgcgagacttgccgaaagcctccccaactgcaagacagtggacatcggcccgggattgttcttgctccaggaagacaacccggaccttatcggcagtgagatcgcgcgc tggctccccgggctggtttaa

Example 6: Use of Labeled Protein in Protein Interaction Reactions

This example demonstrates that protein labeled with benzothiazole dyeconjugates can be used in protein interaction studies. In order toeasily identify proteins that were expressed in the cell free expressionsystems without relying on labeling with a compound of the invention,parallel protein expression reactions were performed where one of thesets of the reactions contained FluoroTect™ Green_(Lys) in vitroTranslation Labeling System (Promega Corp). Proteins expressed withFluoroTect become fluorescently labeled with a dye that is addedinternal to the termini of the protein. The label is detected byexposing a sample of the protein to 488 nm light and detecting emittedlight above 510 nm. Use of this particular second labeling method allowslabeling of a protein by FluoroTect to be easily distinguished from asignal from a compound of the invention used to label an N-terminus of aprotein. A protein labeled at its N-terminus with a compound of theinvention is not appreciably excited by light at 488 nm, but is stronglyexcited by light at 633 nm. On the other hand, the FluoroTect dye is notexcited to an appreciable degree by light at 633 nm but is stronglyexcited by light at 488 nm. Thus, by scanning a sample from thereactions below using separate excitation wavelengths of 488 nm and 633nm, one can distinguish protein species made in the in vitro proteinsynthesis reaction from those labeled using the compound of theinvention.

Three 250 μL translation reactions were performed by adding 20 μg of theindicated DNAs to reactions using SP6 TnT High Yield Extract (PromegaCorp., Madison, Wis.) assembled in the reactions as recommended by thesupplier. The three constructs added encoded: 1) a fusion proteinbetween HaloTag and the catalytic subunit of protein kinase A; 2) aconstruct expressing a metal binding peptide followed by a TEV cleavagesite followed by a Cysteine residue and the regulatory subunit ofprotein kinase A (also known as RI alpha), and; 3) a third reactionidentical to number two, but also containing 10 μL of FluoroTect(Promega Corp). The reactions were allowed to incubate at 25° C. for 120minutes.

A 225 μL sample of the three translation reactions were processedthrough microbiospin columns (BioRad) according to the manufacturersrecommendations following the 120 minute incubation. After microbiospinprocessing, 12 μL of 20×ProTEV buffer, 2.4 μL of 0.1M DTT, and ˜10 UProTEV protease (Promega Corp.) were added and the tubes incubated for60 minutes at room temperature. After TEV treatment, 30 μL MagneHis wasused to remove the protease. To the processed lysate was added 2.8 μL of2 mM TCEP and 3.8 μL of 125 μM PBI 3168 and the tubes were incubated for60 minutes at room temperature. Finally, freshly reduced cysteine wasadded to these tubes to react with any excess benzothiazole reagent.

A 400 μL sample of HaloLink Magnetic Beads (Promega Corporation Catalog#G9311) was washed and resuspended in 300 μL as per the manufacturersrecommendation, then 50 μL of slurry was used to capture HaloTag fusionproteins from 50 μL of SP6 translation reactions, again as described bythe manufacturer. Other HaloLink resins such as Promega Cat. #G1911 orCat. #G1912 can also be used. See M. Urh et al., “Halolink™ Resin ForProtein Pull-Down And Analysis” Cell Notes 2006, 14, 15-19. Afterwashing, the resin was resuspended in kinase buffer (40 mM Tris-HCl, pH7.5, 20 mM MgCl₂, 0.1 mg/mL BSA) and the resin was incubated with thecyanobenzothiazole labeled prey proteins. After these incubations andwashing with 1× wash buffer, samples of the protein solutions werefractionated on SDS PAGE gels, analyzed by SDS PAGE electrophoresis, andthe gels were imaged using excitation with 633 nm laser.

Visualization of the image produced by scanning with a lazar fordetection of the Fluorotect product showed specific protein pull-down(FIG. 8B). Scanning the gels of FIG. 8A and FIG. 8B with a 633 nm lasershows the detection of the pulldown with a redcyanobenzothiazole-derived dye. More interaction was observed with thesample versus the control resin, which demonstrates that the label canbe used to detect protein:protein interactions.

FIG. 8A, scanned at a wavelength that showed only the redcyanobenzothiazole dye label, shows pulldown scanning for fluorescenceof PBI 3168 (prepared from Alexa 633 SE and the corresponding primaryamine linked to a cyanobenzothiazole; releases light fluorescently afterexcitation by light of 633 nm). In FIG. 8B, the same gel, was scannedfor Fluorotect. All are pulldowns; bait and prey are as follows:

Lanes 1+4, Bait=buffer, Prey=RI-alpha.

Lanes 2+5, Bait=TNT High Yield Lysate (no translation), Prey=RI-alpha.

Lanes 3+6, Bait is HaloTag-PKA, Prey=RI-alpha.

Samples in lanes 1-3 were Fluorotect labeled during translation and 4-6were not. The far red (633 nm) was used because of its spectralseparation from Fluorotect so no crosstalk between dyes was observed. InFIG. 8B, a doublet of RI-alpha can be seen because a small amount wasnot cleaved by TEV. The faint band much higher on the gel is likely anoligomer and is also present in FIG. 8A. While there is somenon-specifically labeled species in this cyanobenzothiazole-labeledexample, the ability to use the cyanobenzothiazole-labeling method todetect protein:protein interactions is clearly demonstrated.

Scanning the gel with the 488 nm or 523 nm lasers allowed specific pulldown of RI alpha by Immobilized PKA. Much less capture of this proteinwas seen if it was added to the control resin, thus confirming thatthese results were being produced as a result of the well knowninteraction between PKA and the regulatory subunit of PKA.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A method for immobilizing a protein of interest,comprising: a) contacting a sample comprising an N-terminal cysteinelabeled protein of interest with a cyanobenzothiazole derivative ofFormula I

wherein Z is H, F, Cl, Br, I, CN, amino, alkylamino, dialkylamino, alkylester, carboxy, carboxylic acid salt, alkyl amide, phosphate, alkylphosphonate, sulfate, alkyl sulfonate, nitro, or C₁-C₁₀ alkyl optionallyunsaturated and optionally substituted with amino, hydroxy, oxo (═O),nitro, thiol, or halo; each R¹ is independently H, F, Cl, Br, I, CN,C₁-C₆ alkyl, C₁-C₆ alkoxy, or C₁-C₆ alkylthio, wherein each alkyl,alkoxy, or alkylthio is optionally substituted with F, Cl, Br, I, amino,alkenyl, alkynyl, cycloalkyl, aryl, alkyl sulfonate, or CO₂M wherein Mis H, an organic cation, or an inorganic cation; n is 0, 1, or 2; Y is alinking group comprising C₁-C₁₆ alkylene optionally substituted with oneor more halo, oxo (═O), C_(r)O₆alkyl, or C₁-C₆alkoxy, and optionallyinterrupted with one or more N(R¹), O, S, or —NH—C(═O)— groups; and X isa solid support; and b) immobilizing the protein of interest.
 2. Themethod of claim 1, wherein the protein of interest is purified.
 3. Themethod of claim 1, wherein the solid support is a particle, resin, bead,or surface.
 4. The method of claim 1, wherein the solid supportcomprises a silicate, polymer, sepharose, cellulose, alginate,polystyrene, a membrane, or glass.
 5. The method of claim 1, wherein thesolid support is paramagnetic or magnetic.
 6. The method of claim 1,wherein the protein of interest maintains its functionality uponimmobilization.
 7. The method of claim 1, wherein immobilizationprovides site-specific immobilization of the protein of interest.
 8. Themethod of claim 7, wherein the site-specific immobilization occurs atthe N-terminal cysteine of the protein of interest.